RNA INTERFERENCE MEDIATED INHIBITION OF STROMAL CELL-DERIVED FACTOR-1 (SDF-1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

ABSTRACT

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of stromal cell-derived factor-1 (SDF-1) gene expression and/or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in SDF-1 gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating or that mediate RNA interference (RNAi) against SDF-1 gene expression. Such small nucleic acid molecules are useful, for example, in providing compositions for treatment of traits, diseases and conditions that can respond to modulation of SDF-1 expression in a subject, such as ocular disease, cancer and proliferative diseases and any other disease, condition, trait or indication that can respond to the level of SDF-1 in a cell or tissue.

This application is a continuation of U.S. patent application Ser. No.11/140,328, filed May 27, 2005, which is a continuation-in-part of U.S.patent application Ser. No. 10/923,536, filed Aug. 20, 2004, which is acontinuation-in-part of International Patent Application No.PCT/US04/16390, filed May 24, 2004, which is a continuation-in-part ofU.S. patent application Ser. No. 10/826,966, filed Apr. 16, 2004 (nowabandoned), which is continuation-in-part of U.S. patent applicationSer. No. 10/757,803, filed Jan. 14, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/720,448,filed Nov. 24, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/693,059, filed Oct. 23, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 10/444,853,filed May 23, 2003, which is a continuation-in-part of InternationalPatent Application No. PCT/US03/05346, filed Feb. 20, 2003, and acontinuation-in-part of International Patent Application No.PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit ofU.S. Provisional Application No. 60/358,580, filed Feb. 20, 2002, U.S.Provisional Application No. 60/363,124, filed Mar. 11, 2002, U.S.Provisional Application No. 60/386,782, filed Jun. 6, 2002, U.S.Provisional Application No. 60/406,784, filed Aug. 29, 2002, U.S.Provisional Application No. 60/408,378, filed Sep. 5, 2002, U.S.Provisional Application No. 60/409,293, filed Sep. 9, 2002, and U.S.Provisional Application No. 60/440,129, filed Jan. 15, 2003. The parentU.S. patent application Ser. No. 11/140,328, filed May 27, 2005 is alsoa continuation-in-part of International Patent Application No.PCT/US05/04270, filed Feb. 9, 2005, which claims the benefit of U.S.Provisional Application No. 60/543,480, filed Feb. 10, 2004. The instantapplication claims the benefit of all the listed applications, which arehereby incorporated by reference herein in their entireties, includingthe drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing55USCNT”,created on Jul. 8, 2008, which is 350,824 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of stromal cell-derivedfactor-1 (SDF-1) gene expression and/or activity. The present inventionis also directed to compounds, compositions, and methods relating totraits, diseases and conditions that respond to the modulation ofexpression and/or activity of genes involved in SDF-1 gene expressionpathways or other cellular processes that mediate the maintenance ordevelopment of such traits, diseases and conditions. Specifically, theinvention relates to small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules capable of mediating or that mediate RNA interference(RNAi) against SDF-1 gene expression. Such small nucleic acid moleculesare useful, for example, in providing compositions for treatment oftraits, diseases and conditions that can respond to modulation of SDF-1expression in a subject, such as ocular disease, cancer andproliferative diseases and any other disease, condition, trait orindication that can respond to the level of SDF-1 in a cell or tissue.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the SDF-1 DNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the SDF-1 DNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of a siRNA duplexis required for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al, International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certain singlestranded siRNA constructs, including certain 5′-phosphorylated singlestranded siRNAs that mediate RNA interference in Hela cells. Harborth etal., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105,describe certain chemically and structurally modified siRNA molecules.Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically andstructurally modified siRNA molecules. Woolf et al., International PCTPublication Nos. WO 03/064626 and WO 03/064625 describe certainchemically modified dsRNA constructs. Butler et al., 2005, J. Clin.Invest., 115, 86-93 describe prevention of retinal neovascularization ina murine model of proliferative retinopathy using antibodies that blockSDF-1.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating stromal cell-derived factor-1 (SDF-1) gene expressionusing short interfering nucleic acid (siNA) molecules. This inventionalso relates to compounds, compositions, and methods useful formodulating the expression and activity of other genes involved inpathways of SDF-1 gene expression and/or activity by RNA interference(RNAi) using small nucleic acid molecules. In particular, the instantinvention features small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules and methods used to modulate the expression SDF-1genes.

A siNA of the invention can be unmodified or chemically-modified. A siNAof the instant invention can be chemically synthesized, expressed from avector or enzymatically synthesized. The instant invention also featuresvarious chemically-modified synthetic short interfering nucleic acid(siNA) molecules capable of modulating target gene expression oractivity in cells by RNA interference (RNAi). The use ofchemically-modified siNA improves various properties of native siNAmolecules through increased resistance to nuclease degradation in vivoand/or through improved cellular uptake. Further, contrary to earlierpublished studies, siNA having multiple chemical modifications retainsits RNAi activity. The siNA molecules of the instant invention provideuseful reagents and methods for a variety of therapeutic, cosmetic,veterinary, diagnostic, target validation, genomic discovery, geneticengineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofSDF-1 genes encoding proteins, such as proteins comprising stromalcell-derived factor-1 associated with the maintenance and/or developmentof proliferative retinopathy (e.g., diabetic retinopathy) in a subjector organism such as genes encoding sequences comprising those sequencesreferred to by GenBank Accession Nos. shown in Table I, referred toherein generally as stromal cell-derived factor-1, SDF-1, or CXCL12a.The description below of the various aspects and embodiments of theinvention is provided with reference to exemplary SDF-1 gene. However,the various aspects and embodiments are also directed to other stromalcell-derived factor genes, such as stromal cell-derived factor homologgenes and transcript variants and polymorphisms (e.g., single nucleotidepolymorphism, (SNPs)) associated with certain stromal cell-derivedfactor genes. As such, the various aspects and embodiments are alsodirected to other genes that are involved in stromal cell-derived factormediated pathways of signal transduction or gene expression that areinvolved, for example, in the maintenance and/or development ofconditions or disease states described herein in a subject or organism.These additional genes can be analyzed for target sites using themethods described for stromal cell-derived factor genes herein. Thus,the modulation of other genes and the effects of such modulation of theother genes can be performed, determined, and measured as describedherein.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, wherein saidsiNA molecule comprises about 15 to about 28 base pairs.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of aSDF-1 RNA, wherein said siNA molecule comprises about 15 to about 28base pairs.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of aSDF-1 RNA via RNA interference (RNAi), wherein the double stranded siNAmolecule comprises a first and a second strand, each strand of the siNAmolecule is about 18 to about 28 nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the SDF-1 RNA for the siNA molecule to directcleavage of the SDF-1 RNA via RNA interference, and the second strand ofsaid siNA molecule comprises nucleotide sequence that is complementaryto the first strand.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of aSDF-1 RNA via RNA interference (RNAi), wherein the double stranded siNAmolecule comprises a first and a second strand, each strand of the siNAmolecule is about 18 to about 23 nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the SDF-1 RNA for the siNA molecule to directcleavage of the SDF-1 RNA via RNA interference, and the second strand ofsaid siNA molecule comprises nucleotide sequence that is complementaryto the first strand.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a SDF-1 RNA via RNA interference (RNAi), whereineach strand of the siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the SDF-1 RNA for the siNAmolecule to direct cleavage of the SDF-1 RNA via RNA interference.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a SDF-1 RNA via RNA interference (RNAi), whereineach strand of the siNA molecule is about 18 to about 23 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the SDF-1 RNA for the siNAmolecule to direct cleavage of the SDF-1 RNA via RNA interference.

In one embodiment, the invention features a siNA molecule thatdown-regulates expression of a SDF-1 gene or that directs cleavage of aSDF-1 RNA, for example, wherein the SDF-1 gene or RNA comprises proteinencoding sequence. In one embodiment, the invention features a siNAmolecule that down-regulates expression of a SDF-1 gene or that directscleavage of a SDF-1 RNA, for example, wherein the SDF-1 gene or RNAcomprises non-coding sequence or regulatory elements involved in SDF-1gene expression (e.g., non-coding RNA).

In one embodiment, a siNA of the invention is used to inhibit theexpression of SDF-1 genes or a SDF-1 gene family, wherein the genes orgene family sequences share sequence homology. Such homologous sequencescan be identified as is known in the art, for example using sequencealignments. siNA molecules can be designed to target such homologoussequences, for example using perfectly complementary sequences or byincorporating non-canonical base pairs, for example mismatches and/orwobble base pairs, that can provide additional target sequences. Ininstances where mismatches are identified, non-canonical base pairs (forexample, mismatches and/or wobble bases) can be used to generate siNAmolecules that target more than one gene sequence. In a non-limitingexample, non-canonical base pairs such as UU and CC base pairs are usedto generate siNA molecules that are capable of targeting sequences fordiffering polynucleotide targets that share sequence homology. As such,one advantage of using siNAs of the invention is that a single siNA canbe designed to include nucleic acid sequence that is complementary tothe nucleotide sequence that is conserved between the homologous genes.In this approach, a single siNA can be used to inhibit expression ofmore than one gene instead of using more than one siNA molecule totarget the different genes.

In one embodiment, the invention features a siNA molecule having RNAiactivity against SDF-1 RNA, wherein the siNA molecule comprises asequence complementary to any RNA having SDF-1 encoding sequence, suchas those sequences having GenBank Accession Nos. shown in Table I. Inanother embodiment, the invention features a siNA molecule having RNAiactivity against SDF-1 RNA, wherein the siNA molecule comprises asequence complementary to an RNA having variant SDF-1 encoding sequence,for example other mutant SDF-1 genes not shown in Table I but known inthe art to be associated with the maintenance and/or development ofdiseases and disorders in a subject or organism (e.g., proliferativeretinopathy). Chemical modifications as shown in Tables III and IV orotherwise described herein can be applied to any siNA construct of theinvention. In another embodiment, a siNA molecule of the inventionincludes a nucleotide sequence that can interact with nucleotidesequence of a SDF-1 gene and thereby mediate silencing of SDF-1 geneexpression, for example, wherein the siNA mediates regulation of SDF-1gene expression by cellular processes that modulate the chromatinstructure or methylation patterns of the SDF-1 gene and preventtranscription of the SDF-1 gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of proteins arising from SDF-1haplotype polymorphisms that are associated with a trait, disease orcondition such as ocular disease (e.g., proliferative retinopathy) in asubject or organism. Analysis of genes, or protein or RNA levels can beused to identify subjects with such polymorphisms or those subjects whoare at risk of developing traits, conditions, or diseases describedherein. These subjects are amenable to treatment, for example, treatmentwith siNA molecules of the invention and any other composition useful intreating diseases related to SDF-1 gene expression. As such, analysis ofSDF-1 protein or RNA levels can be used to determine treatment type andthe course of therapy in treating a subject. Monitoring of SDF-1 proteinor RNA levels can be used to predict treatment outcome and to determinethe efficacy of compounds and compositions that modulate the leveland/or activity of certain SDF-1 proteins associated with a trait,condition, or disease.

In one embodiment of the invention a siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a SDF-1 protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a SDF-1 gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a SDF-1 protein or a portion thereof. The siNAmolecule further comprises a sense region, wherein said sense regioncomprises a nucleotide sequence of a SDF-1 gene or a portion thereof.

In another embodiment, the invention features a siNA molecule comprisingnucleotide sequence, for example, nucleotide sequence in the antisenseregion of the siNA molecule that is complementary to a nucleotidesequence or portion of sequence of a SDF-1 gene. In another embodiment,the invention features a siNA molecule comprising a region, for example,the antisense region of the siNA construct, complementary to a sequencecomprising a SDF-1 gene sequence or a portion thereof.

In yet another embodiment, the invention features a siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in U.S. Ser. No.10/923,536 and PCT/US03/05028, both incorporated by reference herein.

In one embodiment, the antisense region of siNA constructs comprises asequence complementary to sequence having any of target SEQ ID NOs.shown in Tables II and III. In one embodiment, the antisense region ofsiNA constructs of the invention constructs comprises sequence havingany of antisense (lower) SEQ ID NOs. in Tables II and III and FIGS. 4and 5. In another embodiment, the sense region of siNA constructs of theinvention comprises sequence having any of sense (upper) SEQ ID NOs. inTables II and III and FIGS. 4 and 5.

In one embodiment, a siNA molecule of the invention comprises any of SEQID NOs. 1-646. The sequences shown in SEQ ID NOs: 1-646 are notlimiting. A siNA molecule of the invention can comprise any contiguousSDF-1 sequence (e.g., about 15 to about 25 or more, or about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous SDF-1 nucleotides).

In yet another embodiment, the invention features a siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention a siNA molecule comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a SDF-1 RNA sequence ora portion thereof, and wherein said siNA further comprises a sensestrand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and wherein saidsense strand and said antisense strand are distinct nucleotide sequenceswhere at least about 15 nucleotides in each strand are complementary tothe other strand.

In another embodiment of the invention a siNA molecule of the inventioncomprises an antisense region having about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein the antisense region is complementary to a SDF-1DNA sequence, and wherein said siNA further comprises a sense regionhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein said senseregion and said antisense region are comprised in a linear moleculewhere the sense region comprises at least about 15 nucleotides that arecomplementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a stromal cell-derivedfactor (SDF) gene. Because SDF genes can share some degree of sequencehomology with each other, siNA molecules can be designed to target aclass of SDF genes or alternately specific SDF genes (e.g., polymorphicvariants) by selecting sequences that are either shared amongstdifferent SDF targets or alternatively that are unique for a specificSDF target. Therefore, in one embodiment, the siNA molecule can bedesigned to target conserved regions of SDF RNA sequences havinghomology among several SDF gene variants so as to target a class of SDFgenes with one siNA molecule. Accordingly, in one embodiment, the siNAmolecule of the invention modulates the expression of one or both SDF-1alleles in a subject. In another embodiment, the siNA molecule can bedesigned to target a sequence that is unique to a specific SDF-1 RNAsequence (e.g., a single SDF-1 allele or SDF-1 single nucleotidepolymorphism (SNP)) due to the high degree of specificity that the siNAmolecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 15 to about 30 base pairs between oligonucleotidescomprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2,or 3) nucleotides, for example, about 21-nucleotide duplexes with about19 base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs. In yet another embodiment, siNA molecules ofthe invention comprise duplex nucleic acid molecules with blunt ends,where both ends are blunt, or alternatively, where one of the ends isblunt.

In one embodiment, the invention features one or morechemically-modified siNA constructs having specificity for targetnucleic acid molecules, such as DNA, or RNA encoding a protein ornon-coding RNA associated with the expression of SDF-1 genes. In oneembodiment, the invention features a RNA based siNA molecule (e.g., asiNA comprising 2′-OH nucleotides) having specificity for nucleic acidmolecules that includes one or more chemical modifications describedherein. Non-limiting examples of such chemical modifications includewithout limitation phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethylnucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides,2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No.10/981,966 filed Nov. 5, 2004, incorporated by reference herein),“universal base” nucleotides, “acyclic” nucleotides, 5-C-methylnucleotides, and terminal glyceryl and/or inverted deoxy abasic residueincorporation. These chemical modifications, when used in various siNAconstructs, (e.g., RNA based siNA constructs), are shown to preserveRNAi activity in cells while at the same time, dramatically increasingthe serum stability of these compounds.

In one embodiment, a siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, a siNAmolecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, a siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single stranded, the percent modification can be based uponthe total number of nucleotides present in the single stranded siNAmolecules Likewise, if the siNA molecule is double stranded, the percentmodification can be based upon the total number of nucleotides presentin the sense strand, antisense strand, or both the sense and antisensestrands.

A siNA molecule of the invention can comprise modified nucleotides atvarious locations within the siNA molecule. In one embodiment, a doublestranded siNA molecule of the invention comprises modified nucleotidesat internal base paired positions within the siNA duplex. For example,internal positions can comprise positions from about 3 to about 19nucleotides from the 5′-end of either sense or antisense strand orregion of a 21 nucleotide siNA duplex having 19 base pairs and twonucleotide 3′-overhangs. In another embodiment, a double stranded siNAmolecule of the invention comprises modified nucleotides at non-basepaired or overhang regions of the siNA molecule. For example, overhangpositions can comprise positions from about 20 to about 21 nucleotidesfrom the 5′-end of either sense or antisense strand or region of a 21nucleotide siNA duplex having 19 base pairs and two nucleotide3′-overhangs. In another embodiment, a double stranded siNA molecule ofthe invention comprises modified nucleotides at terminal positions ofthe siNA molecule. For example, such terminal regions include the3′-position, 5′-position, for both 3′ and 5′-positions of the senseand/or antisense strand or region of the siNA molecule. In anotherembodiment, a double stranded siNA molecule of the invention comprisesmodified nucleotides at base-paired or internal positions, non-basepaired or overhang regions, and/or terminal regions, or any combinationthereof.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a SDF-1gene or that directs cleavage of a SDF-1 RNA. In one embodiment, thedouble stranded siNA molecule comprises one or more chemicalmodifications and each strand of the double-stranded siNA is about 21nucleotides long. In one embodiment, the double-stranded siNA moleculedoes not contain any ribonucleotides. In another embodiment, thedouble-stranded siNA molecule comprises one or more ribonucleotides. Inone embodiment, each strand of the double-stranded siNA moleculeindependently comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein each strand comprises about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotidesthat are complementary to the nucleotides of the other strand. In oneembodiment, one of the strands of the double-stranded siNA moleculecomprises a nucleotide sequence that is complementary to a nucleotidesequence or a portion thereof of the SDF-1 gene, and the second strandof the double-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the SDF-1 gene or aportion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, comprising anantisense region, wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence of the SDF-1gene or a portion thereof, and a sense region, wherein the sense regioncomprises a nucleotide sequence substantially similar to the nucleotidesequence of the SDF-1 gene or a portion thereof. In one embodiment, theantisense region and the sense region independently comprise about 15 toabout 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30) nucleotides, wherein the antisense region comprises about15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotidesof the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, comprising asense region and an antisense region, wherein the antisense regioncomprises a nucleotide sequence that is complementary to a nucleotidesequence of RNA encoded by the SDF-1 gene or a portion thereof and thesense region comprises a nucleotide sequence that is complementary tothe antisense region.

In one embodiment, a siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, a siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 34” or “Stab 3F”-“Stab 34F” (Table IV) or anycombination thereof (see Table IV)) and/or any length described hereincan comprise blunt ends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, a siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 15 to about 30nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides). Other nucleotides present in a bluntended siNA molecule can comprise, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the siNAmolecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a doublestranded siNA molecule having no overhanging nucleotides. The twostrands of a double stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. The sense regioncan be connected to the antisense region via a linker molecule, such asa polynucleotide linker or a non-nucleotide linker.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, wherein thesiNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, andwherein each strand of the siNA molecule comprises one or more chemicalmodifications. In another embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of a SDF-1 gene or a portionthereof, and the second strand of the double-stranded siNA moleculecomprises a nucleotide sequence substantially similar to the nucleotidesequence or a portion thereof of the SDF-1 gene. In another embodiment,one of the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of aSDF-1 gene or portion thereof, and the second strand of thedouble-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or portion thereof ofthe SDF-1 gene. In another embodiment, each strand of the siNA moleculecomprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strandcomprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that arecomplementary to the nucleotides of the other strand. The SDF-1 gene cancomprise, for example, sequences referred to herein or incorporatedherein by reference.

In one embodiment, a siNA molecule of the invention comprises noribonucleotides. In another embodiment, a siNA molecule of the inventioncomprises ribonucleotides.

In one embodiment, a siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a SDF-1 gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the SDF-1 gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides andthe antisense region comprises at least about 15 to about 30 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.The SDF-1 gene can comprise, for example, sequences referred to hereinor incorporated by reference herein. In another embodiment, the siNA isa double stranded nucleic acid molecule, where each of the two strandsof the siNA molecule independently comprise about 15 to about 40 (e.g.about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, and where one ofthe strands of the siNA molecule comprises at least about 15 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) nucleotides thatare complementary to the nucleic acid sequence of the SDF-1 gene or aportion thereof.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a SDF-1 gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In one embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. The SDF-1 genecan comprise, for example, sequences referred herein or incorporated byreference herein

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, comprising asense region and an antisense region, wherein the antisense regioncomprises a nucleotide sequence that is complementary to a nucleotidesequence of RNA encoded by the SDF-1 gene or a portion thereof and thesense region comprises a nucleotide sequence that is complementary tothe antisense region, and wherein the siNA molecule has one or moremodified pyrimidine and/or purine nucleotides. In one embodiment, thepyrimidine nucleotides in the sense region are 2′-O-methyl pyrimidinenucleotides or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purinenucleotides present in the sense region are 2′-deoxy purine nucleotides.In another embodiment, the pyrimidine nucleotides in the sense regionare 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-O-methyl purine nucleotides. Inanother embodiment, the pyrimidine nucleotides in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides. In oneembodiment, the pyrimidine nucleotides in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the antisense region are 2′-O-methyl or 2′-deoxy purinenucleotides. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of thesense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule, and wherein thefragment comprising the sense region includes a terminal cap moiety atthe 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the fragment.In one embodiment, the terminal cap moiety is an inverted deoxy abasicmoiety or glyceryl moiety. In one embodiment, each of the two fragmentsof the siNA molecule independently comprise about 15 to about 30 (e.g.about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In another embodiment, each of the two fragments of thesiNA molecule independently comprise about 15 to about 40 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23,33, 34, 35, 36, 37, 38, 39, or 40) nucleotides. In a non-limitingexample, each of the two fragments of the siNA molecule comprise about21 nucleotides.

In one embodiment, the invention features a siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide, 2′-O-trifluoromethyl nucleotide,2′-O-ethyl-trifluoromethoxy nucleotide, or 2′-O-difluoromethoxy-ethoxynucleotide or any other modified nucleoside/nucleotide described in U.S.Ser. No. 10/981,966 filed Nov. 5, 2004, incorporated by referenceherein. The siNA can be, for example, about 15 to about 40 nucleotidesin length. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy, 4′-thiopyrimidine nucleotides. In one embodiment, the modified nucleotides inthe siNA include at least one 2′-deoxy-2′-fluoro cytidine or2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, themodified nucleotides in the siNA include at least one 2′-fluoro cytidineand at least one 2′-deoxy-2′-fluoro uridine nucleotides. In oneembodiment, all uridine nucleotides present in the siNA are2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as phosphorothioate linkage. Inone embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a method of increasing thestability of a siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all uridine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, allcytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as a phosphorothioate linkage.In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, comprising asense region and an antisense region, wherein the antisense regioncomprises a nucleotide sequence that is complementary to a nucleotidesequence of RNA encoded by the SDF-1 gene or a portion thereof and thesense region comprises a nucleotide sequence that is complementary tothe antisense region, and wherein the purine nucleotides present in theantisense region comprise 2′-deoxy- purine nucleotides. In analternative embodiment, the purine nucleotides present in the antisenseregion comprise 2′-O-methyl purine nucleotides. In either of the aboveembodiments, the antisense region can comprise a phosphorothioateinternucleotide linkage at the 3′ end of the antisense region.Alternatively, in either of the above embodiments, the antisense regioncan comprise a glyceryl modification at the 3′ end of the antisenseregion. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of theantisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of a siNA molecule of theinvention comprises sequence complementary to a portion of an endogenoustranscript having sequence unique to a particular disease or traitrelated allele in a subject or organism, such as sequence comprising asingle nucleotide polymorphism (SNP) associated with the disease ortrait specific allele (see for example Haines et al., Mar. 10, 2005,Science Express, 1110359, describing Complement factor H polymorphismsassociated with age related macular degeneration, “AMD”). As such, theantisense region of a siNA molecule of the invention can comprisesequence complementary to sequences that are unique to a particularallele to provide specificity in mediating selective RNAi against thedisease, condition, or trait related allele. In one embodiment, a siNAmolecule of the invention comprises sequence complementary to complementfactor H sequence polymorphism (e.g., Genbank Accession No.NM_(—)001014975 or NM_(—)000186) rather than SDF-1 sequence. Such siNAmolecules can be designed to target complement factor H sequence as isdescribed for SDF-1 siNA molecules herein.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SDF-1 gene or that directs cleavage of a SDF-1 RNA, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acidmolecule, where each strand is about 21 nucleotides long and where about19 nucleotides of each fragment of the siNA molecule are base-paired tothe complementary nucleotides of the other fragment of the siNAmolecule, wherein at least two 3′ terminal nucleotides of each fragmentof the siNA molecule are not base-paired to the nucleotides of the otherfragment of the siNA molecule. In another embodiment, the siNA moleculeis a double stranded nucleic acid molecule, where each strand is about19 nucleotide long and where the nucleotides of each fragment of thesiNA molecule are base-paired to the complementary nucleotides of theother fragment of the siNA molecule to form at least about 15 (e.g., 15,16, 17, 18, or 19) base pairs, wherein one or both ends of the siNAmolecule are blunt ends. In one embodiment, each of the two 3′ terminalnucleotides of each fragment of the siNA molecule is a2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In anotherembodiment, all nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule. In another embodiment, the siNA molecule is a doublestranded nucleic acid molecule of about 19 to about 25 base pairs havinga sense region and an antisense region, where about 19 nucleotides ofthe antisense region are base-paired to the nucleotide sequence or aportion thereof of the RNA encoded by the SDF-1 gene. In anotherembodiment, about 21 nucleotides of the antisense region are base-pairedto the nucleotide sequence or a portion thereof of the RNA encoded bythe SDF-1 gene. In any of the above embodiments, the 5′-end of thefragment comprising said antisense region can optionally include aphosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa SDF-1 RNA sequence, wherein the siNA molecule does not contain anyribonucleotides and wherein each strand of the double-stranded siNAmolecule is about 15 to about 30 nucleotides. In one embodiment, thesiNA molecule is 21 nucleotides in length. Examples ofnon-ribonucleotide containing siNA constructs are combinations ofstabilization chemistries shown in Table I in any combination ofSense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14,15, 17, 18, 19, 20, or 32 sense or antisense strands or any combinationthereof). Herein, numeric Stab chemistries can include both 2′-fluoroand 2′-OCF3 versions of the chemistries shown in Table I. For example,“Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In oneembodiment, the invention features a chemically synthesized doublestranded RNA molecule that directs cleavage of a SDF-1 RNA via RNAinterference, wherein each strand of said RNA molecule is about 15 toabout 30 nucleotides in length; one strand of the RNA molecule comprisesnucleotide sequence having sufficient complementarity to the SDF-1 RNAfor the RNA molecule to direct cleavage of the SDF-1 RNA via RNAinterference; and wherein at least one strand of the RNA moleculeoptionally comprises one or more chemically modified nucleotidesdescribed herein, such as without limitation deoxynucleotides,2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides,2′-O-methoxyethyl nucleotides, 4′-thio nucleotides, 2′-O-trifluoromethylnucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides,2′-O-difluoromethoxy-ethoxy nucleotides, etc.

In one embodiment, a SDF-1 RNA of the invention comprises sequenceencoding a protein.

In one embodiment, SDF-1 RNA of the invention comprises non-coding RNAsequence (e.g., miRNA, snRNA siRNA etc.).

In one embodiment, the invention features a medicament comprising a siNAmolecule of the invention.

In one embodiment, the invention features an active ingredientcomprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to inhibit,down-regulate, or reduce expression of a SDF-1 gene, wherein the siNAmolecule comprises one or more chemical modifications and each strand ofthe double-stranded siNA is independently about 15 to about 30 or more(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 or more) nucleotides long. In one embodiment, the siNA molecule ofthe invention is a double stranded nucleic acid molecule comprising oneor more chemical modifications, where each of the two fragments of thesiNA molecule independently comprise about 15 to about 40 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23,33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of thestrands comprises at least 15 nucleotides that are complementary tonucleotide sequence of target encoding RNA or a portion thereof. In anon-limiting example, each of the two fragments of the siNA moleculecomprise about 21 nucleotides. In another embodiment, the siNA moleculeis a double stranded nucleic acid molecule comprising one or morechemical modifications, where each strand is about 21 nucleotide longand where about 19 nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule, wherein at least two 3′ terminal nucleotides of eachfragment of the siNA molecule are not base-paired to the nucleotides ofthe other fragment of the siNA molecule. In another embodiment, the siNAmolecule is a double stranded nucleic acid molecule comprising one ormore chemical modifications, where each strand is about 19 nucleotidelong and where the nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or19) base pairs, wherein one or both ends of the siNA molecule are bluntends. In one embodiment, each of the two 3′ terminal nucleotides of eachfragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, suchas a 2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region and comprising one or more chemical modifications,where about 19 nucleotides of the antisense region are base-paired tothe nucleotide sequence or a portion thereof of the RNA encoded by theSDF-1 gene. In another embodiment, about 21 nucleotides of the antisenseregion are base-paired to the nucleotide sequence or a portion thereofof the RNA encoded by the SDF-1 gene. In any of the above embodiments,the 5′-end of the fragment comprising said antisense region canoptionally include a phosphate group.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits,down-regulates, or reduces expression of a SDF-1 gene, wherein one ofthe strands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of SDF-1 RNA or a portion thereof, the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a SDF-1 gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofSDF-1 RNA or a portion thereof, wherein the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a SDF-1 gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofSDF-1 RNA that encodes a protein or portion thereof, the other strand isa sense strand which comprises nucleotide sequence that is complementaryto a nucleotide sequence of the antisense strand and wherein a majorityof the pyrimidine nucleotides present in the double-stranded siNAmolecule comprises a sugar modification. In one embodiment, each strandof the siNA molecule comprises about 15 to about 30 or more (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ormore) nucleotides, wherein each strand comprises at least about 15nucleotides that are complementary to the nucleotides of the otherstrand. In one embodiment, the siNA molecule is assembled from twooligonucleotide fragments, wherein one fragment comprises the nucleotidesequence of the antisense strand of the siNA molecule and a secondfragment comprises nucleotide sequence of the sense region of the siNAmolecule. In one embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In still another embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-deoxy purine nucleotides. Inanother embodiment, the antisense strand comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-O-methyl purine nucleotides. In a further embodiment the sensestrand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety(e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotidemoiety such as inverted thymidine) is present at the 5′-end, the 3′-end,or both of the 5′ and 3′ ends of the sense strand. In anotherembodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aSDF-1 gene, wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, eachof the two strands of the siNA molecule can comprise about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule. In another embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule, wherein at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In anotherembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine.In one embodiment, each strand of the siNA molecule is base-paired tothe complementary nucleotides of the other strand of the siNA molecule.In one embodiment, about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of theantisense strand are base-paired to the nucleotide sequence of the SDF-1RNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g.,about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisensestrand are base-paired to the nucleotide sequence of the SDF-1 RNA or aportion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aSDF-1 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of SDF-1 RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the 5′-end of the antisense strand optionally includes aphosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aSDF-1 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of SDF-1 RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the untranslatedregion or a portion thereof of the SDF-1 RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aSDF-1 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of SDF-1 RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand, wherein a majority of the pyrimidine nucleotidespresent in the double-stranded siNA molecule comprises a sugarmodification, and wherein the nucleotide sequence of the antisensestrand is complementary to a nucleotide sequence of the SDF-1 RNA or aportion thereof that is present in the SDF-1 RNA.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of a siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of a siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a SDF-1 coding or non-coding RNA or DNAsequence and the sense region can comprise sequence complementary to theantisense region. The siNA molecule can comprise two distinct strandshaving complementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbonemodified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified, each X and Y is independently O, S, N, alkyl, orsubstituted alkyl, each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl andwherein W, X, Y, and Z are optionally not all O. In another embodiment,a backbone modification of the invention comprises a phosphonoacetateand/or thiophosphonoacetate internucleotide linkage (see for exampleSheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, a siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotideshaving Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to SDF-1RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to SDF-1 RNA. In one embodiment, R3 and/or R7comprises a conjugate moiety and a linker (e.g., a nucleotide ornon-nucleotide linker as described herein or otherwise known in theart). Non-limiting examples of conjugate moieties include ligands forcellular receptors, such as peptides derived from naturally occurringprotein ligands; protein localization sequences, including cellular ZIPcode sequences; antibodies; nucleic acid aptamers; vitamins and otherco-factors, such as folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; steroids, andpolyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotideshaving Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to SDF-1 RNA or a non-nucleosidic base such as phenyl,naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to SDF-1 RNA. In one embodiment,R3 and/or R7 comprises a conjugate moiety and a linker (e.g., anucleotide or non-nucleotide linker as described herein or otherwiseknown in the art). Non-limiting examples of conjugate moieties includeligands for cellular receptors, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a 5′-terminal phosphategroup having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features a siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to aSDF-1 RNA, wherein the siNA molecule comprises an all RNA siNA molecule.In another embodiment, the invention features a siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of a siNA molecule of the invention,for example a siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more phosphorothioateinternucleotide linkages. For example, in a non-limiting example, theinvention features a chemically-modified short interfering nucleic acid(siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in one siNA strand. In yet another embodiment,the invention features a chemically-modified short interfering nucleicacid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or morephosphorothioate internucleotide linkages in both siNA strands. Thephosphorothioate internucleotide linkages can be present in one or botholigonucleotide strands of the siNA duplex, for example in the sensestrand, the antisense strand, or both strands. The siNA molecules of theinvention can comprise one or more phosphorothioate internucleotidelinkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand, the antisense strand, or both strands. For example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) consecutivephosphorothioate internucleotide linkages at the 5′-end of the sensestrand, the antisense strand, or both strands. In another non-limitingexample, an exemplary siNA molecule of the invention can comprise one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy and/or aboutone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy,2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In another embodiment, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides ofthe sense and/or antisense siNA strand are chemically-modified with2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In another embodiment, the invention features a siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, or more) universal base modified nucleotides, and optionallya terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and5′-ends of the sense strand; and wherein the antisense strand comprisesabout 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides,and optionally a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the antisense strand. In anotherembodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more, pyrimidine nucleotides of the sense and/or antisense siNAstrand are chemically-modified with 2′-deoxy, 2′-O-methyl,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orabout one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy,2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In another embodiment, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides ofthe sense and/or antisense siNA strand are chemically-modified with2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, forexample, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends, being present in the same ordifferent strand.

In another embodiment, the invention features a siNA molecule, whereinthe sense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides,and optionally a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the antisense strand. In anotherembodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more pyrimidine nucleotides of the sense and/or antisense siNA strandare chemically-modified with 2′-deoxy, 2′-O-methyl,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5, for example about 1, 2,3, 4, 5 or more phosphorothioate internucleotide linkages and/or aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) canbe at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one orboth siNA sequence strands. In addition, the 2′-5′ internucleotidelinkage(s) can be present at various other positions within one or bothsiNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a pyrimidinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein one or both strands of the siNA molecule can comprise a 2′-5′internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex hasabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemicalmodification comprises a structure having any of Formulae I-VII. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a duplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, a siNA molecule of the invention comprises a single strandedhairpin structure, wherein the siNA is about 36 to about 70 (e.g., about36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) base pairs, and wherein the siNA can include achemical modification comprising a structure having any of FormulaeI-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a linearoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the linear oligonucleotide forms a hairpin structurehaving about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphategroup that can be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, andwherein the siNA can include one or more chemical modificationscomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 25 toabout 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)nucleotides that is chemically-modified with one or more chemicalmodifications having any of Formulae I-VII or any combination thereof,wherein the linear oligonucleotide forms an asymmetric hairpin structurehaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a5′-terminal phosphate group that can be chemically modified as describedherein (for example a 5′-terminal phosphate group having Formula IV). Inone embodiment, an asymmetric hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. In another embodiment, an asymmetric hairpinsiNA molecule of the invention comprises a loop portion comprising anon-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, whereinthe sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides in length, wherein the sense region and the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprisesan asymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23)nucleotides in length and wherein the sense region is about 3 to about15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification, which comprises a structure having anyof Formulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a circularoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the circular oligonucleotide forms a dumbbell shapedstructure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2. In one embodiment,R3 and/or R7 comprises a conjugate moiety and a linker (e.g., anucleotide or non-nucleotide linker as described herein or otherwiseknown in the art). Non-limiting examples of conjugate moieties includeligands for cellular receptors, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spermine or spermidine.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3,R8 or R13 serve as points of attachment to the siNA molecule of theinvention. In one embodiment, R3 and/or R7 comprises a conjugate moietyand a linker (e.g., a nucleotide or non-nucleotide linker as describedherein or otherwise known in the art). Non-limiting examples ofconjugate moieties include ligands for cellular receptors, such aspeptides derived from naturally occurring protein ligands; proteinlocalization sequences, including cellular ZIP code sequences;antibodies; nucleic acid aptamers; vitamins and other co-factors, suchas folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; steroids, andpolyamines, such as PEI, spermine or spermidine.

In another embodiment, a siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention. In one embodiment, R3 and/or R1 comprises aconjugate moiety and a linker (e.g., a nucleotide or non-nucleotidelinker as described herein or otherwise known in the art). Non-limitingexamples of conjugate moieties include ligands for cellular receptors,such as peptides derived from naturally occurring protein ligands;protein localization sequences, including cellular ZIP code sequences;antibodies; nucleic acid aptamers; vitamins and other co-factors, suchas folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; steroids, andpolyamines, such as PEI, spermine or spermidine.

By “ZIP code” sequences is meant, any peptide or protein sequence thatis involved in cellular topogenic signaling mediated transport (see forexample Ray et al., 2004, Science, 306(1501): 1505).

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises Oand is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside ornon-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of theinvention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends ofa siNA molecule of the invention. For example, chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) can be present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the antisense strand, the sense strand, or both antisense andsense strands of the siNA molecule. In one embodiment, the chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) is present at the 5′-end and 3′-end of the sense strand andthe 3′-end of the antisense strand of a double stranded siNA molecule ofthe invention. In one embodiment, the chemically modified nucleoside ornon-nucleoside (e.g., a moiety having Formula V, VI or VII) is presentat the terminal position of the 5′-end and 3′-end of the sense strandand the 3′-end of the antisense strand of a double stranded siNAmolecule of the invention. In one embodiment, the chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the two terminal positions of the 5′-end and 3′-endof the sense strand and the 3′-end of the antisense strand of a doublestranded siNA molecule of the invention. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the penultimate position of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double stranded siNA molecule of the invention. In addition,a moiety having Formula VII can be present at the 3′-end or the 5′-endof a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a 3′-3′, 3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 4′-thionucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), wherein anynucleotides comprising a 3′-terminal nucleotide overhang that arepresent in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the senseregion are 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g.,one or more or all) purine nucleotides present in the sense region are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein anynucleotides comprising a 3′-terminal nucleotide overhang that arepresent in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the antisenseregion are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g.,one or more or all) purine nucleotides present in the antisense regionare 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein anynucleotides comprising a 3′-terminal nucleotide overhang that arepresent in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the antisenseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the antisenseregion are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) inside a cell or reconstituted invitro system comprising a sense region, wherein one or more pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or morepurine nucleotides present in the sense region are 2′-deoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides), and an antisense region, wherein one ormore pyrimidine nucleotides present in the antisense region are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and one or more purine nucleotides present in theantisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides). The sense region and/orthe antisense region can have a terminal cap modification, such as anymodification described herein or shown in FIG. 10, that is optionallypresent at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of thesense and/or antisense sequence. The sense and/or antisense region canoptionally further comprise a 3′-terminal nucleotide overhang havingabout 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. Theoverhang nucleotides can further comprise one or more (e.g., about 1, 2,3, 4 or more) phosphorothioate, phosphonoacetate, and/orthiophosphonoacetate internucleotide linkages. Non-limiting examples ofthese chemically-modified siNAs are shown in FIGS. 4 and 5 and Table Iherein. In any of these described embodiments, the purine nucleotidespresent in the sense region are alternatively 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides) and one or morepurine nucleotides present in the antisense region are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Also, in any ofthese embodiments, one or more purine nucleotides present in the senseregion are alternatively purine ribonucleotides (e.g., wherein allpurine nucleotides are purine ribonucleotides or alternately a pluralityof purine nucleotides are purine ribonucleotides) and any purinenucleotides present in the antisense region are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Additionally, in anyof these embodiments, one or more purine nucleotides present in thesense region and/or present in the antisense region are alternativelyselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides and 2′-O-methyl nucleotides (e.g., wherein all purinenucleotides are selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides and 2′-O-methyl nucleotides or alternately a plurality ofpurine nucleotides are selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides, 4′-thio nucleotides and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxy abasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a conjugate covalentlyattached to the chemically-modified siNA molecule. Non-limiting examplesof conjugates contemplated by the invention include conjugates andligands described in Vargeese et al., U.S. Ser. No. 10/427,160, filedApr. 30, 2003, incorporated by reference herein in its entirety,including the drawings. In another embodiment, the conjugate iscovalently attached to the chemically-modified siNA molecule via abiodegradable linker. In one embodiment, the conjugate molecule isattached at the 3′-end of either the sense strand, the antisense strand,or both strands of the chemically-modified siNA molecule. In anotherembodiment, the conjugate molecule is attached at the 5′-end of eitherthe sense strand, the antisense strand, or both strands of thechemically-modified siNA molecule. In yet another embodiment, theconjugate molecule is attached both the 3′-end and 5′-end of either thesense strand, the antisense strand, or both strands of thechemically-modified siNA molecule, or any combination thereof. In oneembodiment, a conjugate molecule of the invention comprises a moleculethat facilitates delivery of a chemically-modified siNA molecule into abiological system, such as a cell. In another embodiment, the conjugatemolecule attached to the chemically-modified siNA molecule is a ligandfor a cellular receptor, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spermine or spermidine. Examplesof specific conjugate molecules contemplated by the instant inventionthat can be attached to chemically-modified siNA molecules are describedin Vargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotide,non-nucleotide, or mixed nucleotide/non-nucleotide linker is used, forexample, to attach a conjugate moiety to the siNA. In one embodiment, anucleotide linker of the invention can be a linker of ≧2 nucleotides inlength, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides inlength. In another embodiment, the nucleotide linker can be a nucleicacid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein ismeant a nucleic acid molecule that binds specifically to a targetmolecule wherein the nucleic acid molecule has sequence that comprises asequence recognized by the target molecule in its natural setting.Alternately, an aptamer can be a nucleic acid molecule that binds to atarget molecule where the target molecule does not naturally bind to anucleic acid. The target molecule can be any molecule of interest. Forexample, the aptamer can be used to bind to a ligand-binding domain of aprotein, thereby preventing interaction of the naturally occurringligand with the protein. This is a non-limiting example and those in theart will recognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, a siNA molecule can beassembled from a single oligonculeotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, a siNA molecule can beassembled from a single oligonculeotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presense of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence. In anotherembodiment, the single stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group. In another embodiment, the singlestranded siNA molecule of the invention comprises a 5′-terminalphosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclicphosphate). In another embodiment, the single stranded siNA molecule ofthe invention comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. Inyet another embodiment, the single stranded siNA molecule of theinvention comprises one or more chemically modified nucleotides ornon-nucleotides described herein. For example, all the positions withinthe siNA molecule can include chemically-modified nucleotides such asnucleotides having any of Formulae I-VII, or any combination thereof tothe extent that the ability of the siNA molecule to support RNAiactivity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence, wherein one or morepyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein anypurine nucleotides present in the antisense region are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and a terminal capmodification, such as any modification described herein or shown in FIG.10, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence. The siNA optionally furthercomprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more)terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, whereinthe terminal nucleotides can further comprise one or more (e.g., 1, 2,3, 4 or more) phosphorothioate, phosphonoacetate, and/orthiophosphonoacetate internucleotide linkages, and wherein the siNAoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group. In any of these embodiments, any purinenucleotides present in the antisense region are alternatively 2′-deoxypurine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxypurine nucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides). Also, in any of these embodiments, anypurine nucleotides present in the siNA (i.e., purine nucleotides presentin the sense and/or antisense region) can alternatively be lockednucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides areLNA nucleotides or alternately a plurality of purine nucleotides are LNAnucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA are alternatively 2′-methoxyethyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-methoxyethyl purinenucleotides or alternately a plurality of purine nucleotides are2′-methoxyethyl purine nucleotides). In another embodiment, any modifiednucleotides present in the single stranded siNA molecules of theinvention comprise modified nucleotides having properties orcharacteristics similar to naturally occurring ribonucleotides. Forexample, the invention features siNA molecules including modifiednucleotides having a Northern conformation (e.g., Northernpseudorotation cycle, see for example Saenger, Principles of NucleicAcid Structure, Springer-Verlag ed., 1984). As such, chemically modifiednucleotides present in the single stranded siNA molecules of theinvention are preferably resistant to nuclease degradation while at thesame time maintaining the capacity to mediate RNAi.

In one embodiment, a siNA molecule of the invention comprises chemicallymodified nucleotides or non-nucleotides (e.g., having any of FormulaeI-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides) at alternatingpositions within one or more strands or regions of the siNA molecule.For example, such chemical modifications can be introduced at everyother position of a RNA based siNA molecule, starting at either thefirst or second nucleotide from the 3′-end or 5′-end of the siNA. In anon-limiting example, a double stranded siNA molecule of the inventionin which each strand of the siNA is 21 nucleotides in length is featuredwherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of eachstrand are chemically modified (e.g., with compounds having any ofFormulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In anothernon-limiting example, a double stranded siNA molecule of the inventionin which each strand of the siNA is 21 nucleotides in length is featuredwherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strandare chemically modified (e.g., with compounds having any of FormulaeI-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). Such siNAmolecules can further comprise terminal cap moieties and/or backbonemodifications as described herein.

In one embodiment, the invention features a composition comprising oneor more siNA molecules of the instant invention and one or more siNAmolecules targeting VEGF (e.g., VEGF-A, VEGF-B, VEGF-C, or VEGF-D)and/or VEGFR (e.g., VEGFR1, VEGFR2, or VEGFR3), (see for example U.S.Ser. Nos. 10/962,898, 10/944,644, and 10/844,076, all incorporated byreference herein).

In one embodiment, the invention features a composition comprising oneor more siNA molecules of the instant invention and one or more siNAmolecules targeting placental derived growth factor (PGF), (see forexample U.S. Ser. No. 10/922,761, incorporated by reference herein).

In one embodiment, the invention features a composition comprising oneor more siNA molecules of the instant invention and one or more siNAmolecules targeting hypoxia induced growth factor (HIF-1), (see forexample U.S. Ser. No. 10/922,554, incorporated by reference herein).

In one embodiment, the invention features a composition comprising oneor more siNA molecules of the instant invention and one or more siNAmolecules targeting Angiopoietin (e.g., ANG1, ANG2, ANG3 and/or ANG4),(see for example U.S. Ser. No. 10/922,626, incorporated by referenceherein).

In one embodiment, the invention features a composition comprising oneor more siNA molecules of the instant invention and one or more siNAmolecules targeting Endothelial Cell Growth Factor (e.g., ECGF1), (seefor example U.S. Ser. No. 10/922,034, incorporated by reference herein).

In one embodiment, the invention features a composition comprising oneor more siNA molecules of the instant invention and one or more siNAmolecules targeting complement factor H (e.g., siNA molecules targetingcomplement factor H polymorphisms Genbank Accession No. NM_(—)001014975or NM_(—)000186, see for example Haines et al., Mar. 10, 2005, ScienceExpress, 1110359).

In one embodiment, the invention features a method for modulating theexpression of a SDF-1 gene within a cell comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified orunmodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the SDF-1 gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate (e.g.,inhibit) the expression of the SDF-1 gene in the cell.

In one embodiment, the invention features a method for modulating theexpression of a first SDF-1 gene and a second gene within a cellcomprising: (a) synthesizing a first siNA molecule, which can bechemically-modified or unmodified as described herein, wherein one ofthe siNA strands comprises a sequence complementary to RNA of the SDF-1gene; and (b) synthesizing a second siNA molecule, which can bechemically-modified or unmodified as described herein, wherein one ofthe siNA strands comprises a sequence complementary to RNA of the secondgene; and (c) introducing the first and second siNA molecules into acell under conditions suitable to modulate (e.g., inhibit) theexpression of the first SDF-1 gene and the second gene in the cell. Inanother embodiment, the second gene comprises a vascular endothelialgrowth factor (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), vascularendothelial growth factor receptor (e.g., VEGFR1, VEGFR2, and/orVEGFR3), hypoxia induced growth factor (e.g., HIF-1), Angiopoietin(e.g., ANG1, ANG2, ANG3 and/or ANG4), Endothelial Cell Growth Factor(e.g., ECGF1), placental derived growth factor (PGF), and/or complementfactor H gene.

In one embodiment, the invention features a method for modulating theexpression of a SDF-1 gene within a cell comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified orunmodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the SDF-1 gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequence of the SDF-1 RNA; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate (e.g.,inhibit) the expression of the SDF-1 gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one SDF-1 gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified or unmodified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the SDF-1 genes; and (b)introducing the siNA molecules into a cell under conditions suitable tomodulate (e.g., inhibit) the expression of the SDF-1 genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more SDF-1 genes within a cell comprising: (a)synthesizing one or more siNA molecules of the invention, which can bechemically-modified or unmodified, wherein the siNA strands comprisesequences complementary to RNA of the SDF-1 genes and wherein the sensestrand sequences of the siNAs comprise sequences identical orsubstantially similar to the sequences of the SDF-1 RNAs; and (b)introducing the siNA molecules into a cell under conditions suitable tomodulate (e.g., inhibit) the expression of the SDF-1 genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients.

In one embodiment, the invention features a method of modulating theexpression of a SDF-1 gene in a tissue explant comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SDF-1 gene; and (b) introducing thesiNA molecule into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate (e.g.,inhibit) the expression of the SDF-1 gene in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate (e.g., inhibit)the expression of the SDF-1 gene in that organism.

In one embodiment, the invention features a method for modulating theexpression of a first SDF-1 gene and a second gene in a tissue explantcomprising: (a) synthesizing a first siNA molecule, which can bechemically-modified or unmodified as described herein, wherein one ofthe siNA strands comprises a sequence complementary to RNA of the SDF-1gene; and (b) synthesizing a second siNA molecule, which can bechemically-modified or unmodified as described herein, wherein one ofthe siNA strands comprises a sequence complementary to RNA of the secondgene; and (c) introducing the first and second siNA molecules into thetissue explant under conditions suitable to modulate (e.g., inhibit) theexpression of the first SDF-1 gene and the second gene in the tissueexplant. In another embodiment, the second gene comprises a vascularendothelial growth factor (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D),vascular endothelial growth factor receptor (e.g., VEGFR1, VEGFR2,and/or VEGFR3), hypoxia induced growth factor (e.g., HIF-1),Angiopoietin (e.g., ANG1, ANG2, ANG3 and/or ANG4), Endothelial CellGrowth Factor (e.g., ECGF1), placental derived growth factor (PGF),and/or complement factor H gene. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate (e.g., inhibit) the expression of the SDF-1 geneand the second gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a SDF-1 gene in a tissue explant comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SDF-1 gene and wherein the sensestrand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequence of the SDF-1 RNA; and (b)introducing the siNA molecule into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate (e.g.,inhibit) the expression of the SDF-1 gene in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate (e.g., inhibit)the expression of the SDF-1 gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one SDF-1 gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SDF-1 genes; and (b) introducingthe siNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate (e.g.,inhibit) the expression of the SDF-1 genes in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate (e.g., inhibit)the expression of the SDF-1 genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a SDF-1 gene in a subject or organism comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SDF-1 gene; and (b) introducing thesiNA molecule into the subject or organism under conditions suitable tomodulate (e.g., inhibit) the expression of the SDF-1 gene in the subjector organism. The level of target protein or RNA can be determined usingvarious methods well-known in the art.

In one embodiment, the invention features a method for modulating theexpression of a first SDF-1 gene and a second gene in a subject ororganism comprising: (a) synthesizing a first siNA molecule, which canbe chemically-modified or unmodified as described herein, wherein one ofthe siNA strands comprises a sequence complementary to RNA of the SDF-1gene; and (b) synthesizing a second siNA molecule, which can bechemically-modified or unmodified as described herein, wherein one ofthe siNA strands comprises a sequence complementary to RNA of the secondgene; and (c) introducing the first and second siNA molecules into thesubject or organism under conditions suitable to modulate (e.g.,inhibit) the expression of the first SDF-1 gene and the second gene inthe subject or organism. In another embodiment, the second genecomprises a vascular endothelial growth factor (e.g., VEGF-A, VEGF-B,VEGF-C, and/or VEGF-D), vascular endothelial growth factor receptor(e.g., VEGFR1, VEGFR2, and/or VEGFR3), hypoxia induced growth factor(e.g., HIF-1), Angiopoietin (e.g., ANG1, ANG2, ANG3 and/or ANG4),Endothelial Cell Growth Factor (e.g., ECGF1), placental derived growthfactor (PGF), and/or complement factor H gene.

In another embodiment, the invention features a method of modulating theexpression of more than one SDF-1 gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SDF-1 genes; and (b) introducingthe siNA molecules into the subject or organism under conditionssuitable to modulate (e.g., inhibit) the expression of the SDF-1 genesin the subject or organism. The level of target protein or RNA can bedetermined as is known in the art.

In one embodiment, the invention features a method for modulating theexpression of a SDF-1 gene within a cell comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified,wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the SDF-1 gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate (e.g.,inhibit) the expression of the SDF-1 gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one SDF-1 gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the SDF-1 gene; and (b)contacting the cell in vitro or in vivo with the siNA molecule underconditions suitable to modulate (e.g., inhibit) the expression of theSDF-1 genes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a SDF-1 gene in a tissue explant (e.g., a cochlear, skin,heart, liver, spleen, cornea, retina, macula, lung, stomach, kidney,vein, artery, hair, appendage, or limb transplant, or any other organ,tissue or cell as can be transplanted from one organism to another orback to the same organism from which the organ, tissue or cell isderived) comprising: (a) synthesizing a siNA molecule of the invention,which can be chemically-modified, wherein the siNA comprises a singlestranded sequence having complementarity to RNA of the SDF-1 gene; and(b) contacting a cell of the tissue explant derived from a particularsubject or organism with the siNA molecule under conditions suitable tomodulate (e.g., inhibit) the expression of the SDF-1 gene in the tissueexplant. In another embodiment, the method further comprises introducingthe tissue explant back into the subject or organism the tissue wasderived from or into another subject or organism under conditionssuitable to modulate (e.g., inhibit) the expression of the SDF-1 gene inthat subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one SDF-1 gene in a tissue explant (e.g., acochlear, skin, heart, liver, spleen, cornea, retina, macula, lung,stomach, kidney, vein, artery, hair, appendage, or limb transplant, orany other organ, tissue or cell as can be transplanted from one organismto another or back to the same organism from which the organ, tissue orcell is derived) comprising: (a) synthesizing siNA molecules of theinvention, which can be chemically-modified, wherein the siNA comprisesa single stranded sequence having complementarity to RNA of the SDF-1gene; and (b) introducing the siNA molecules into a cell of the tissueexplant derived from a particular subject or organism under conditionssuitable to modulate (e.g., inhibit) the expression of the SDF-1 genesin the tissue explant. In another embodiment, the method furthercomprises introducing the tissue explant back into the subject ororganism the tissue was derived from or into another subject or organismunder conditions suitable to modulate (e.g., inhibit) the expression ofthe SDF-1 genes in that subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a SDF-1 gene in a subject or organism comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the SDF-1 gene; and (b)introducing the siNA molecule into the subject or organism underconditions suitable to modulate (e.g., inhibit) the expression of theSDF-1 gene in the subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one SDF-1 gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the SDF-1 gene; and (b)introducing the siNA molecules into the subject or organism underconditions suitable to modulate (e.g., inhibit) the expression of theSDF-1 genes in the subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a SDF-1 gene in a subject or organism comprisingcontacting the subject or organism with a siNA molecule of the inventionunder conditions suitable to modulate (e.g., inhibit) the expression ofthe SDF-1 gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a disease, disorder, trait or condition related to geneexpression in a subject or organism comprising contacting the subject ororganism with a siNA molecule or composition of the invention underconditions suitable to modulate the expression of the SDF-1 gene and/orother genes in the subject or organism. The reduction of gene expressionand thus reduction in the level of the respective protein/RNA relieves,to some extent, the symptoms of the disease, disorder, trait orcondition.

In one embodiment, the invention features a method for treating orpreventing cancer in a subject or organism comprising contacting thesubject or organism with a siNA molecule or composition of the inventionunder conditions suitable to modulate the expression of the SDF-1 geneand/or other genes in the subject or organism whereby the treatment orprevention of cancer is achieved. In one embodiment, the other gene is avascular endothelial growth factor (e.g., VEGF-A, VEGF-B, VEGF-C, and/orVEGF-D), vascular endothelial growth factor receptor (e.g., VEGFR1,VEGFR2, and/or VEGFR3), hypoxia induced growth factor (e.g., HIF-1),Angiopoietin (e.g., ANG1, ANG2, ANG3 and/or ANG4), Endothelial CellGrowth Factor (e.g., ECGF1), placental derived growth factor (PGF),and/or complement factor H gene.

In one embodiment, the invention features a method for treating orpreventing a proliferative disease or condition in a subject or organismcomprising contacting the subject or organism with a siNA molecule orcomposition of the invention under conditions suitable to modulate theexpression of the SDF-1 gene and/or other genes in the subject ororganism whereby the treatment or prevention of the proliferativedisease or condition is achieved. In one embodiment, the inventionfeatures contacting the subject or organism with a siNA molecule of theinvention via local administration to relevant tissues or cells, such ascells and tissues involved in proliferative disease. In one embodiment,the other gene is a vascular endothelial growth factor (e.g., VEGF-A,VEGF-B, VEGF-C, and/or VEGF-D), vascular endothelial growth factorreceptor (e.g., VEGFR1, VEGFR2, and/or VEGFR3), hypoxia induced growthfactor (e.g., HIF-1), Angiopoietin (e.g., ANG1, ANG2, ANG3 and/or ANG4),Endothelial Cell Growth Factor (e.g., ECGF1), placental derived growthfactor (PGF), and/or complement factor H gene.

In one embodiment, the invention features a method for treating orpreventing a cardiovascular disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule or composition of the invention under conditions suitableto modulate the expression of the SDF-1 gene and/or other genes in thesubject or organism whereby the treatment or prevention of thecardiovascular disease, disorder, trait or condition is achieved. In oneembodiment, the other gene is a vascular endothelial growth factor(e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), vascular endothelialgrowth factor receptor (e.g., VEGFR1, VEGFR2, and/or VEGFR3), hypoxiainduced growth factor (e.g., HIF-1), Angiopoietin (e.g., ANG1, ANG2,ANG3 and/or ANG4), Endothelial Cell Growth Factor (e.g., ECGF1),placental derived growth factor (PGF), and/or complement factor H gene.

In one embodiment, the invention features a method for treating orpreventing a respiratory disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule or composition of the invention under conditions suitableto modulate the expression of the SDF-1 gene and/or other genes in thesubject or organism whereby the treatment or prevention of therespiratory disease, disorder, trait or condition is achieved. In oneembodiment, the other gene is a vascular endothelial growth factor(e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), vascular endothelialgrowth factor receptor (e.g., VEGFR1, VEGFR2, and/or VEGFR3), hypoxiainduced growth factor (e.g., HIF-1), Angiopoietin (e.g., ANG1, ANG2,ANG3 and/or ANG4), Endothelial Cell Growth Factor (e.g., ECGF1),placental derived growth factor (PGF), and/or complement factor H gene.

In one embodiment, the invention features a method for treating orpreventing an ocular disease, disorder, trait or condition in a subjector organism comprising contacting the subject or organism with a siNAmolecule or composition of the invention under conditions suitable tomodulate the expression of the SDF-1 gene and/or other genes in thesubject or organism whereby the treatment or prevention of the oculardisease, disorder, trait or condition is achieved. In one embodiment,the other gene is a vascular endothelial growth factor (e.g., VEGF-A,VEGF-B, VEGF-C, and/or VEGF-D), vascular endothelial growth factorreceptor (e.g., VEGFR1, VEGFR2, and/or VEGFR3), hypoxia induced growthfactor (e.g., HIF-1), Angiopoietin (e.g., ANG1, ANG2, ANG3 and/or ANG4),Endothelial Cell Growth Factor (e.g., ECGF1), placental derived growthfactor (PGF), and/or complement factor H gene.

In one embodiment, the invention features a method for treating orpreventing a kidney/renal disease, disorder, trait or condition (e.g.,polycystic kidney disease etc.) in a subject or organism comprisingcontacting the subject or organism with a siNA molecule or compositionof the invention under conditions suitable to modulate the expression ofthe SDF-1 gene and/or other genes in the subject or organism whereby thetreatment or prevention of the kidney/renal disease, disorder, trait orcondition is achieved. In one embodiment, the other gene is a vascularendothelial growth factor (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D),vascular endothelial growth factor receptor (e.g., VEGFR1, VEGFR2,and/or VEGFR3), hypoxia induced growth factor (e.g., HIF-1),Angiopoietin (e.g., ANG1, ANG2, ANG3 and/or ANG4), Endothelial CellGrowth Factor (e.g., ECGF1), placental derived growth factor (PGF),and/or complement factor H gene.

In one embodiment, the invention features contacting the subject ororganism with a siNA molecule or composition of the invention via localadministration to relevant tissues or cells, such as cells and tissuesassociated with a disease, trait, or condition. In one embodiment, theinvention features contacting the subject or organism with a siNAmolecule or composition of the invention via systemic administration(such as via intravenous or subcutaneous administration of siNA) torelevant tissues or cells, such as tissues or cells involved in themaintenance or development of a disease, trait, or condition in asubject or organism. The siNA molecule or composition of the inventioncan be formulated or conjugated as described herein or otherwise knownin the art to target appropriate tissues or cells in the subject ororganism.

In any of the methods of treatment of the invention, the siNA orcomposition can be administered to the subject as a course of treatment,for example administration at various time intervals, such as once perday over the course of treatment, once every two days over the course oftreatment, once every three days over the course of treatment, onceevery four days over the course of treatment, once every five days overthe course of treatment, once every six days over the course oftreatment, once per week over the course of treatment, once every otherweek over the course of treatment, once per month over the course oftreatment, etc. In one embodiment, the course of treatment is from aboutone to about 52 weeks or longer (e.g., indefinitely). In one embodiment,the course of treatment is from about one to about 48 months or longer(e.g., indefinitely).

In any of the methods of treatment of the invention, the siNA orcomposition can be administered to the subject systemically as describedherein or otherwise known in the art. Systemic administration caninclude, for example, intravenous, subcutaneous, intramuscular,catheterization, nasopharangeal, transdermal, or gastrointestinaladministration as is generally known in the art.

In one embodiment, in any of the methods of treatment or prevention ofthe invention, the siNA or composition can be administered to thesubject locally or to local tissues as described herein or otherwiseknown in the art. Local administration can include, for example,catheterization, implantation, direct injection (e.g., intraocularinjection), dermal/transdermal application, stenting, ear/eye drops, orportal vein administration to relevant tissues, or any other localadministration technique, method or procedure, as is generally known inthe art.

In another embodiment, the invention features a method of modulating theexpression of more than one SDF-1 gene in a subject or organismcomprising contacting the subject or organism with one or more siNAmolecules or compositions of the invention under conditions suitable tomodulate (e.g., inhibit) the expression of the SDF-1 and/or other genesin the subject or organism.

The siNA molecules of the invention can be designed to down regulate orinhibit gene expression through RNAi targeting of a variety of nucleicacid molecules. In one embodiment, the siNA molecules of the inventionare used to target various DNA corresponding to a target gene, forexample via heterochromatic silencing. In one embodiment, the siNAmolecules of the invention are used to target various RNAs correspondingto a target gene, for example via RNA target cleavage or translationalinhibition. Non-limiting examples of such RNAs include messenger RNA(mRNA), non-coding RNA or regulatory elements, alternate RNA splicevariants of target gene(s), post-transcriptionally modified RNA oftarget gene(s), pre-mRNA of target gene(s), and/or RNA templates. Ifalternate splicing produces a family of transcripts that aredistinguished by usage of appropriate exons, the instant invention canbe used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, cosmetic applications,veterinary applications, pharmaceutical discovery applications,molecular diagnostic and gene function applications, and gene mapping,for example using single nucleotide polymorphism mapping with siNAmolecules of the invention. Such applications can be implemented usingknown gene sequences or from partial sequences available from anexpressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as gene families having homologous sequences. As such,siNA molecules targeting multiple gene or RNA targets can provideincreased therapeutic effect. In one embodiment, the invention featuresthe targeting (cleavage or inhibition of expression or function) of morethan one target gene sequence using a single siNA molecule, by targetingthe conserved sequences of the targeted gene(s).

In addition, siNA can be used to characterize pathways of gene functionin a variety of applications. For example, the present invention can beused to inhibit the activity of target gene(s) in a pathway to determinethe function of uncharacterized gene(s) in gene function analysis, mRNAfunction analysis, or translational analysis. The invention can be usedto determine potential target gene pathways involved in various diseasesand conditions toward pharmaceutical development. The invention can beused to understand pathways of gene expression involved in, for example,the progression and/or maintenance of diseases, traits, and conditionsassociated with SDF-1 gene expression or activity in a subject ororganism.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession, for example, SDF-1 genes encoding RNAsequence(s) referred to herein by Genbank Accession number, for example,Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the SDF-1 RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described herein. In another embodiment, the assaycan comprise a cell culture system in which SDF-1 RNA is expressed. Inanother embodiment, fragments of SDF-1 RNA are analyzed for detectablelevels of cleavage, for example by gel electrophoresis, northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the SDF-1 RNA sequence. The SDF-1 RNA sequence canbe obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siNA construct strands(eg. for a siNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target SDF-1 RNA sequence. Inanother embodiment, the siNA molecules of (a) have strands of a fixedlength, for example about 23 nucleotides in length. In yet anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described in Example 6 herein. In anotherembodiment, the assay can comprise a cell culture system in which SDF-1RNA is expressed. In another embodiment, fragments of SDF-1 RNA areanalyzed for detectable levels of cleavage, for example, by gelelectrophoresis, northern blot analysis, or RNAse protection assays, todetermine the most suitable target site(s) within the target SDF-1 RNAsequence. The target SDF-1 RNA sequence can be obtained as is known inthe art, for example, by cloning and/or transcription for in vitrosystems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a SDF-1 gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the SDF-1 RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides in length. In one embodiment, the assay cancomprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich SDF-1 RNA is expressed. Fragments of SDF-1 RNA are analyzed fordetectable levels of cleavage, for example by gel electrophoresis,northern blot analysis, or RNAse protection assays, to determine themost suitable target site(s) within the SDF-1 RNA sequence. The SDF-1RNA sequence can be obtained as is known in the art, for example, bycloning and/or transcription for in vitro systems, and by expression inin vivo systems.

By “target site” is meant a sequence within a SDF-1 or other target RNAthat is “targeted” for cleavage mediated by a siNA construct whichcontains sequences within its antisense region that are complementary tothe target sequence.

By “detectable level of cleavage” is meant cleavage of SDF-1 RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the SDF-1 RNA. Production of cleavage products from 1-5%of the SDF-1 RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising oneor more siNA molecules of the invention, which can bechemically-modified, in a pharmaceutically acceptable carrier ordiluent. In another embodiment, the invention features a pharmaceuticalcomposition comprising siNA molecules of the invention, which can bechemically-modified, targeting one or more genes in a pharmaceuticallyacceptable carrier or diluent. In another embodiment, the inventionfeatures a method for diagnosing a disease, trait, or condition in asubject comprising administering to the subject a composition of theinvention under conditions suitable for the diagnosis of the disease,trait, or condition in the subject. In another embodiment, the inventionfeatures a method for treating or preventing a disease, trait, orcondition, such as hearing loss, deafness, tinnitus, and/or motion andbalance disorders in a subject, comprising administering to the subjecta composition of the invention under conditions suitable for thetreatment or prevention of the disease, trait, or condition in thesubject, alone or in conjunction with one or more other therapeuticcompounds.

In another embodiment, the invention features a method for validating aSDF-1 gene target, comprising: (a) synthesizing a siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a SDF-1 gene; (b)introducing the siNA molecule into a cell, tissue, subject, or organismunder conditions suitable for modulating expression of the SDF-1 gene inthe cell, tissue, subject, or organism; and (c) determining the functionof the gene by assaying for any phenotypic change in the cell, tissue,subject, or organism.

In another embodiment, the invention features a method for validating atarget comprising: (a) synthesizing a siNA molecule of the invention,which can be chemically-modified, wherein one of the siNA strandsincludes a sequence complementary to RNA of a SDF-1 gene; (b)introducing the siNA molecule into a biological system under conditionssuitable for modulating expression of the SDF-1 gene in the biologicalsystem; and (c) determining the function of the gene by assaying for anyphenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, subject, or organism, or extract thereof. The term biologicalsystem also includes reconstituted RNAi systems that can be used in anin vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a SDF-1 gene in a biological system,including, for example, in a cell, tissue, subject, or organism. Inanother embodiment, the invention features a kit containing more thanone siNA molecule of the invention, which can be chemically-modified,that can be used to modulate the expression of more than one SDF-1 genein a biological system, including, for example, in a cell, tissue,subject, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically-modified. Inanother embodiment, the cell containing a siNA molecule of the inventionis a mammalian cell. In yet another embodiment, the cell containing asiNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention,which can be chemically-modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing asiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizinga siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example, under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide (e.g., SDF-1 RNA or DNA targets),wherein the siNA construct comprises one or more chemical modifications,for example, one or more chemical modifications having any of FormulaeI-VII or any combination thereof that increases the nuclease resistanceof the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with increased nuclease resistancecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased nuclease resistance.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved toxicologic profiles(e.g., having attenuated or no immunstimulatory properties) comprising(a) introducing nucleotides having any of Formula I-VII (e.g., siNAmotifs referred to in Table I) or any combination thereof into a siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedtoxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA formulations of the invention with improved toxicologic profiles(e.g., having attenuated or no immunstimulatory properties) comprising(a) generating a siNA formulation comprising a siNA molecule of theinvention and a delivery vehicle or delivery particle as describedherein or as otherwise known in the art, and (b) assaying the siNAformulation of step (a) under conditions suitable for isolating siNAformulations having improved toxicologic profiles.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the binding affinity between the sense and antisense strandsof the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoa siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against a SDF-1 polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the binding affinity between the antisense strand of the siNAconstruct and a complementary SDF-1 RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against a SDF-1 polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the binding affinity between the antisense strand of the siNAconstruct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary SDF-1 RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary SDF-1 RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against a SDF-1 polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulate the polymerase activity of a cellular polymerase capable ofgenerating additional endogenous siNA molecules having sequence homologyto the chemically-modified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically-modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically-modified siNAmolecule.

In one embodiment, the invention features chemically-modified siNAconstructs that mediate RNAi against a SDF-1 polynucleotide in a cell,wherein the chemical modifications do not significantly effect theinteraction of siNA with a SDF-1 RNA molecule, DNA molecule and/orproteins or other factors that are essential for RNAi in a manner thatwould decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi specificity against polynucleotidetargets comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi specificity. In one embodiment,improved specificity comprises having reduced off target effectscompared to an unmodified siNA molecule. For example, introduction ofterminal cap moieties at the 3′-end, 5′-end, or both 3′ and 5′-ends ofthe sense strand or region of a siNA molecule of the invention candirect the siNA to have improved specificity by preventing the sensestrand or sense region from acting as a template for RNAi activityagainst a corresponding target having complementarity to the sensestrand or sense region.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against a SDF-1polynucleotide comprising (a) introducing nucleotides having any ofFormula I-VII or any combination thereof into a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against a SDF-1RNA comprising (a) introducing nucleotides having any of Formula I-VIIor any combination thereof into a siNA molecule, and (b) assaying thesiNA molecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity against the SDF-1 RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against a SDF-1DNA comprising (a) introducing nucleotides having any of Formula I-VIIor any combination thereof into a siNA molecule, and (b) assaying thesiNA molecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against a SDF-1 polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the cellular uptake of the siNA construct, such as cholesterolconjugation of the siNA.

In another embodiment, the invention features a method for generatingsiNA molecules against a SDF-1 polynucleotide with improved cellularuptake comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against a SDF-1 polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatincreases the bioavailability of the siNA construct, for example, byattaching polymeric conjugates such as polyethyleneglycol or equivalentconjugates that improve the pharmacokinetics of the siNA construct, orby attaching conjugates that target specific tissue types or cell typesin vivo. Non-limiting examples of such conjugates are described inVargeese et al., U.S. Ser. No. 10/201,394 incorporated by referenceherein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; cholesterol derivatives, polyamines, such asspermine or spermidine; and others.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a SDF-1 RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi. In one embodiment, the first nucleotide sequenceof the siNA is chemically modified as described herein. In oneembodiment, the first nucleotide sequence of the siNA is not modified(e.g., is all RNA).

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a SDF-1 RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. In one embodiment, the first nucleotide sequence of thesiNA is chemically modified as described herein. In one embodiment, thefirst nucleotide sequence of the siNA is not modified (e.g., is allRNA). Such design or modifications are expected to enhance the activityof siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a SDF-1 RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference. In one embodiment, the firstnucleotide sequence of the siNA is chemically modified as describedherein. In one embodiment, the first nucleotide sequence of the siNA isnot modified (e.g., is all RNA).

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a SDF-1 RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a SDF-1 RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a SDF-1 RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of a siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, a siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding SDF-1RNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (seeTable I) wherein the 5′-end and 3′-end of the sense strand of the siNAdo not comprise a hydroxyl group or phosphate group. Herein, numericStab chemistries include both 2′-fluoro and 2′-OCF3 versions of thechemistries shown in Table IV. For example, “Stab 7/8” refers to bothStab 7/8 and Stab 7F/8F etc.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of a siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl, (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”,“Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23,or 24 sense strands) chemistries and variants thereof (see Table I)wherein the 5′-end and 3′-end of the sense strand of the siNA do notcomprise a hydroxyl group or phosphate group. Herein, numeric Stabchemistries include both 2′-fluoro and 2′-OCF3 versions of thechemistries shown in Table IV. For example, “Stab 7/8” refers to bothStab 7/8 and Stab 7F/8F etc.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically modified siNA molecules of step (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter, that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 100 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include a siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4-6, and Table IIherein. For example the siNA can be a double-stranded polynucleotidemolecule comprising self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense region having nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.The siNA can be assembled from two separate oligonucleotides, where onestrand is the sense strand and the other is the antisense strand,wherein the antisense and sense strands are self-complementary (i.e.,each strand comprises nucleotide sequence that is complementary tonucleotide sequence in the other strand; such as where the antisensestrand and sense strand form a duplex or double stranded structure, forexample wherein the double stranded region is about 15 to about 30,e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 base pairs; the antisense strand comprises nucleotide sequencethat is complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof (e.g., about 15 to about 25 or more nucleotides of the siNAmolecule are complementary to the target nucleic acid or a portionthereof). Alternatively, the siNA is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha duplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The siNA can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siNA molecule capable of mediating RNAi. The siNA can alsocomprise a single stranded polynucleotide having nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of aSDF-1 gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a SDF-1 gene in a manner thatcauses inhibition of expression of the SDF-1 gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic modulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation pattern to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237). In another non-limiting example, modulation of geneexpression by siNA molecules of the invention can result from siNAmediated cleavage of RNA (either coding or non-coding RNA) via RISC, oralternately, translational inhibition as is known in the art.

In one embodiment, the term “siNA” refers to a composition comprising aplurality of siNA molecules, that can be the same or different (e.g.,that target differing target sequences, have differing chemicalmodifications and/or differing siNA sequence composition).

In one embodiment, a siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCTApplication No. USO4/16390, filed May 24, 2004).

In one embodiment, a siNA molecule of the invention is a multifunctionalsiNA, (see for example FIGS. 16-21 and Jadhav et al., U.S. Ser. No.60/543,480 filed Feb. 10, 2004 and International PCT Application No.USO4/16390, filed May 24, 2004). In one embodiment, the multifunctionalsiNA of the invention can comprise sequence targeting, for example, twoor more regions of SDF-1 RNA (see for example target sequences in TableII).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g., about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of aRNA molecule or equivalent RNA molecules encoding one or more proteinsor protein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing, such as by alterations in DNA methylation patterns and DNAchromatin structure.

By “gene” or “target DNA”, is meant a nucleic acid that encodes an RNA,for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene can also encode afunctional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporalRNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), shortinterfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA(rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-codingRNAs can serve as target nucleic acid molecules for siNA mediated RNAinterference in modulating the activity of fRNA or ncRNA involved infunctional or regulatory cellular processes. Abberant fRNA or ncRNAactivity leading to disease can therefore be modulated by siNA moleculesof the invention. siNA molecules targeting fRNA and ncRNA can also beused to manipulate or alter the genotype or phenotype of a subject,organism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). The gene can be a gene derivedfrom a cell, an endogenous gene, a transgene, or exogenous genes such asgenes of a pathogen, for example a virus, which is present in the cellafter infection thereof. The cell containing the gene can be derivedfrom or contained in any organism, for example a plant, animal,protozoan, virus, bacterium, or fungus. Non-limiting examples of plantsinclude monocots, dicots, or gymnosperms. Non-limiting examples ofanimals include vertebrates or invertebrates. Non-limiting examples offungi include molds or yeasts. For a review, see for example Snyder andGerstein, 2003, Science, 300, 258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric,CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-iminosymmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, ACamino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AUN1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GAamino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GCcarbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GGcarbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GUimino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3, GAcarbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-05-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “target” as used herein is meant, any target protein, peptide, orpolypeptide, such as encoded by Genbank Accession Nos. shown in U.S.Ser. No. 10/923,536 and PCT/US03/05028, both incorporated by referenceherein, including Genbank Accession Nos. referred to in Table I. Theterm “target” also refers to nucleic acid sequences or targetpolynucleotide sequence encoding any target protein, peptide, orpolypeptide, such as proteins, peptides, or polypeptides encoded bysequences having Genbank Accession Nos. shown in U.S. Ser. No.10/923,536 and PCT/US03/05028. The term “target” is also meant toinclude other sequences, such as differing isoforms, mutant genes,splice variants of target polynucleotides, target polymorphisms, andnon-coding or regulatory polynucleotide sequences. In one embodiment,the term “target” refers to SDF-1 target polypeptide sequences, such asSDF-1 RNA and/or SDF-1 DNA.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of a siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” or “target polynucleotide” is meant any nucleicacid sequence whose expression or activity is to be modulated. Thetarget nucleic acid can be DNA or RNA. In one embodiment, a targetnucleic acid of the invention is SDF-1 RNA or DNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. In one embodiment, a siNA molecule ofthe invention comprises about 15 to about 30 or more (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more)nucleotides that are complementary to one or more target nucleic acidmolecules or a portion thereof.

In one embodiment, siNA molecules of the invention that down regulate orreduce SDF-1 gene expression are used for preventing or treatingdiseases, disorders, conditions, or traits in a subject or organism asdescribed herein or otherwise known in the art.

By “proliferative disease” or “cancer” as used herein is meant, anydisease, condition, trait, genotype or phenotype characterized byunregulated cell growth or replication as is known in the art; includingleukemias, for example, acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia, AIDS related cancers such as Kaposi'ssarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “respiratory disease” is meant, any disease or condition affectingthe respiratory tract, such as asthma, chronic obstructive pulmonarydisease or “COPD”, allergic rhinitis, sinusitis, pulmonaryvasoconstriction, inflammation, allergies, impeded respiration,respiratory distress syndrome, cystic fibrosis, pulmonary hypertension,pulmonary vasoconstriction, emphysema, and any other respiratorydisease, condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “cardiovascular disease” is meant and disease or condition affectingthe heart and vasculature, including but not limited to, coronary heartdisease (CHD), cerebrovascular disease (CVD), aortic stenosis,peripheral vascular disease, atherosclerosis, arteriosclerosis,myocardial infarction (heart attack), cerebrovascular diseases (stroke),transient ischaemic attacks (TIA), angina (stable and unstable), atrialfibrillation, arrhythmia, vavular disease, congestive heart failure,hypercholoesterolemia, type I hyperlipoproteinemia, type IIhyperlipoproteinemia, type III hyperlipoproteinemia, type IVhyperlipoproteinemia, type V hyperlipoproteinemia, secondaryhypertrigliceridemia, and familial lecithin cholesterol acyltransferasedeficiency.

By “ocular disease” as used herein is meant, any disease, condition,trait, genotype or phenotype of the eye and related structures as isknown in the art, such as Cystoid Macular Edema, Asteroid Hyalosis,Pathological Myopia and Posterior Staphyloma, Toxocariasis (Ocular LarvaMigrans), Retinal Vein Occlusion, Posterior Vitreous Detachment,Tractional Retinal Tears, Epiretinal Membrane, Diabetic Retinopathy,Lattice Degeneration, Retinal Vein Occlusion, Retinal Artery Occlusion,Macular Degeneration (e.g., age related macular degeneration such as wetAMD or dry AMD), Toxoplasmosis, Choroidal Melanoma, AcquiredRetinoschisis, Hollenhorst Plaque, Idiopathic Central SerousChorioretinopathy, Macular Hole, Presumed Ocular HistoplasmosisSyndrome, Retinal Macroaneursym, Retinitis Pigmentosa, RetinalDetachment, Hypertensive Retinopathy, Retinal Pigment Epithelium (RPE)Detachment, Papillophlebitis, Ocular Ischemic Syndrome, Coats' Disease,Leber's Miliary Aneurysm, Conjunctival Neoplasms, AllergicConjunctivitis, Vernal Conjunctivitis, Acute Bacterial Conjunctivitis,Allergic Conjunctivitis & Vernal Keratoconjunctivitis, ViralConjunctivitis, Bacterial Conjunctivitis, Chlamydial & GonococcalConjunctivitis, Conjunctival Laceration, Episcleritis, Scleritis,Pingueculitis, Pterygium, Superior Limbic Keratoconjunctivitis (SLK ofTheodore), Toxic Conjunctivitis, Conjunctivitis with Pseudomembrane,Giant Papillary Conjunctivitis, Terrien's Marginal Degeneration,Acanthamoeba Keratitis, Fungal Keratitis, Filamentary Keratitis,Bacterial Keratitis, Keratitis Sicca/Dry Eye Syndrome, BacterialKeratitis, Herpes Simplex Keratitis, Sterile Corneal Infiltrates,Phlyctenulosis, Corneal Abrasion & Recurrent Corneal Erosion, CornealForeign Body, Chemical Burs, Epithelial Basement Membrane Dystrophy(EBMD), Thygeson's Superficial Punctate Keratopathy, Corneal Laceration,Salzmann's Nodular Degeneration, Fuchs' Endothelial Dystrophy,Crystalline Lens Subluxation, Ciliary-Block Glaucoma, Primary Open-AngleGlaucoma, Pigment Dispersion Syndrome and Pigmentary Glaucoma,Pseudoexfoliation Syndrom and Pseudoexfoliative Glaucoma, AnteriorUveitis, Primary Open Angle Glaucoma, Uveitic Glaucoma &Glaucomatocyclitic Crisis, Pigment Dispersion Syndrome & PigmentaryGlaucoma, Acute Angle Closure Glaucoma, Anterior Uveitis, Hyphema, AngleRecession Glaucoma, Lens Induced Glaucoma, Pseudoexfoliation Syndromeand Pseudoexfoliative Glaucoma, Axenfeld-Rieger Syndrome, NeovascularGlaucoma, Pars Planitis, Choroidal Rupture, Duane's Retraction Syndrome,Toxic/Nutritional Optic Neuropathy, Aberrant Regeneration of CranialNerve III, Intracranial Mass Lesions, Carotid-Cavernous Sinus Fistula,Anterior Ischemic Optic Neuropathy, Optic Disc Edema & Papilledema,Cranial Nerve III Palsy, Cranial Nerve IV Palsy, Cranial Nerve VI Palsy,Cranial Nerve VII (Facial Nerve) Palsy, Horner's Syndrome, InternuclearOphthalmoplegia, Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil,Optic Nerve Head Drusen, Demyelinating Optic Neuropathy (Optic Neuritis,Retrobulbar Optic Neuritis), Amaurosis Fugax and Transient IschemicAttack, Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum,Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis,Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal CellCarcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis,Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes SimplexBlepharitis, Orbital Cellulitis, Senile Entropion, and Squamous CellCarcinoma.

In one embodiment of the present invention, each sequence of a siNAmolecule of the invention is independently about 15 to about 30nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inanother embodiment, the siNA duplexes of the invention independentlycomprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In anotherembodiment, one or more strands of the siNA molecule of the inventionindependently comprises about 15 to about 30 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) thatare complementary to a target nucleic acid molecule. In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)base pairs. Exemplary siNA molecules of the invention are shown in TableII and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules and compositions of the invention are added directly,or can be complexed with cationic lipids, packaged within liposomes, orotherwise delivered to target cells or tissues. The nucleic acid ornucleic acid complexes can be locally administered to relevant tissuesex vivo, or in vivo through local delivery to the lung, with or withouttheir incorporation in biopolymers. In particular embodiments, thenucleic acid molecules of the invention comprise sequences shown inTables II-III and/or FIGS. 4-5. Examples of such nucleic acid moleculesconsist essentially of sequences defined in these tables and figures.Furthermore, the chemically modified constructs described in Table I canbe applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules or compositions of this invention. The one ormore siNA molecules or compositions can independently be targeted to thesame or different target sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to forpreventing or treating diseases, disorders, conditions, and traitsdescribed herein or otherwise known in the art, in a subject ororganism.

In one embodiment, the siNA molecules of the invention can beadministered to a subject or can be administered to other appropriatecells evident to those skilled in the art, individually or incombination with one or more drugs under conditions suitable for thetreatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to prevent or treat diseases and conditionsdescribed herein in a subject or organism. For example, the describedmolecules could be used in combination with one or more known compounds,treatments, or procedures to prevent or treat diseases, disorders,conditions, and traits described herein in a subject or organism as areknown in the art.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of a siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms a siNA molecule. Non-limiting examplesof such expression vectors are described in Paul et al., 2002, NatureBiotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology,19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina etal., 2002, Nature Medicine, advance online publicationdoi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for a siNA molecule having complementarity to a RNAmolecule referred to by a Genbank Accession numbers, for example GenbankAccession Nos. shown in Table I, U.S. Ser. No. 10/923,536 andPCT/US03/05028, both incorporated by reference herein.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withSDF-1 RNA molecules and down-regulate gene encoding SDF-1 RNA molecules(for example SDF-1 RNA molecules referred to by Genbank Accessionnumbers herein) are expressed from transcription units inserted into DNAor RNA vectors. The recombinant vectors can be DNA plasmids or viralvectors. siNA expressing viral vectors can be constructed based on, butnot limited to, adeno-associated virus, retrovirus, adenovirus, oralphavirus. The recombinant vectors capable of expressing the siNAmolecules can be delivered as described herein, and persist in targetcells. Alternatively, viral vectors can be used that provide fortransient expression of siNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the siNA molecules bind anddown-regulate gene function or expression via RNA interference (RNAi).Delivery of siNA expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from a subject followed by reintroduction into thesubject, or by any other means that would allow for introduction intothe desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form asiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget (e.g., SDF-1) RNA degradation involved in RNAi. Double-strandedRNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP)from foreign single-stranded RNA, for example viral, transposon, orother exogenous RNA, activates the DICER enzyme that in turn generatessiNA duplexes. Alternately, synthetic or expressed siNA can beintroduced directly into a cell by appropriate means. An active siNAcomplex forms which recognizes a target (e.g., SDF-1) RNA, resulting indegradation of the target (e.g., SDF-1) RNA by the RISC endonucleasecomplex or in the synthesis of additional RNA by RNA-dependent RNApolymerase (RdRP), which can activate DICER and result in additionalsiNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target (e.g., SDF-1) RNA sequence, and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target (e.g., SDF-1) RNA sequence, and wherein all pyrimidinenucleotides that may be present are 2′-deoxy-2′-fluoro modifiednucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the sense and antisensestrand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target(e.g., SDF-1) RNA sequence, and wherein all pyrimidine nucleotides thatmay be present are 2′-deoxy-2′-fluoro modified nucleotides except for (NN) nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target (e.g., SDF-1)RNA sequence, wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target (e.g., SDF-1) RNA sequence,and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target (e.g., SDF-1)RNA sequence, and having one 3′-terminal phosphorothioateinternucleotide linkage and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-deoxy nucleotides except for (NN) nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand. The antisense strand of constructsA-F comprise sequence complementary to any target nucleic acid sequenceof the invention. Furthermore, when a glyceryl moiety (L) is present atthe 3′-end of the antisense strand for any construct shown in FIG. 4A-F, the modified internucleotide linkage is optional.

FIG. 5A-F shows non-limiting examples of specific chemically-modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to an exemplary SDF-1 siNA sequence. Suchchemical modifications can be applied to any target polynucleotidesequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, which is followed by a loop sequence of defined sequence (X),comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in a siNA transcript having specificity for a targetsequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double-stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example, by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, and which is followed by a 3′-restriction site (R2) which isadjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double-stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIG. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc.).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palindrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palindrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of SDF-1 gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifuctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifuctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example, a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC to initiate RNA interferencemediated cleavage of its corresponding target. These design parameterscan include destabilization of each end of the siNA construct (see forexample Schwarz et al., 2003, Cell, 115, 199-208). Such destabilizationcan be accomplished for example by using guanosine-cytidine base pairs,alternate base pairs (e.g., wobbles), or destabilizing chemicallymodified nucleotides at terminal nucleotide positions as is known in theart.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC to initiate RNA interference mediatedcleavage of its corresponding target region. These design parameters caninclude destabilization of each end of the siNA construct (see forexample Schwarz et al., 2003, Cell, 115, 199-208). Such destabilizationcan be accomplished for example by using guanosine-cytidine base pairs,alternate base pairs (e.g., wobbles), or destabilizing chemicallymodified nucleotides at terminal nucleotide positions as is known in theart.

FIG. 22(A-H) shows non-limiting examples of tethered multifunctionalsiNA constructs of the invention. In the examples shown, a linker (e.g.,nucleotide or non-nucleotide linker) connects two siNA regions (e.g.,two sense, two antisense, or alternately a sense and an antisense regiontogether. Separate sense (or sense and antisense) sequencescorresponding to a first target sequence and second target sequence arehybridized to their corresponding sense and/or antisense sequences inthe multifunctional siNA. In addition, various conjugates, ligands,aptamers, polymers or reporter molecules can be attached to the linkerregion for selective or improved delivery and/or pharmacokineticproperties.

FIG. 23 shows a non-limiting example of various dendrimer basedmultifunctional siNA designs.

FIG. 24 shows a non-limiting example of various supramolecularmultifunctional siNA designs.

FIG. 25 shows a non-limiting example of a dicer enabled multifunctionalsiNA design using a 30 nucleotide precursor siNA construct. A 30 basepair duplex is cleaved by Dicer into 22 and 8 base pair products fromeither end (8 b.p. fragments not shown). For ease of presentation theoverhangs generated by dicer are not shown—but can be compensated for.Three targeting sequences are shown. The required sequence identityoverlapped is indicated by grey boxes. The N's of the parent 30 b.p.siNA are suggested sites of 2′-OH positions to enable Dicer cleavage ifthis is tested in stabilized chemistries. Note that processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, butrather produces a series of closely related products (with 22+8 beingthe primary site). Therefore, processing by Dicer will yield a series ofactive siNAs.

FIG. 26 shows a non-limiting example of a dicer enabled multifunctionalsiNA design using a 40 nucleotide precursor siNA construct. A 40 basepair duplex is cleaved by Dicer into 20 base pair products from eitherend. For ease of presentation the overhangs generated by dicer are notshown—but can be compensated for. Four targeting sequences are shown.The target sequences having homology are enclosed by boxes. This designformat can be extended to larger RNAs. If chemically stabilized siNAsare bound by Dicer, then strategically located ribonucleotide linkagescan enable designer cleavage products that permit our more extensiverepertoire of multifunctional designs. For example cleavage products notlimited to the Dicer standard of approximately 22-nucleotides can allowmultifunctional siNA constructs with a target sequence identity overlapranging from, for example, about 3 to about 15 nucleotides.

FIG. 27 shows a non-limiting example of additional multifunctional siNAconstruct designs of the invention. In one example, a conjugate, ligand,aptamer, label, or other moiety is attached to a region of themultifunctional siNA to enable improved delivery or pharmacokineticprofiling.

FIG. 28 shows a non-limiting example of additional multifunctional siNAconstruct designs of the invention. In one example, a conjugate, ligand,aptamer, label, or other moiety is attached to a region of themultifunctional siNA to enable improved delivery or pharmacokineticprofiling.

FIG. 29 shows a non-limiting example of a cholesterol linkedphosphoramidite that can be used to synthesize cholesterol conjugatedsiNA molecules of the invention. An example is shown with thecholesterol moiety linked to the 5′-end of the sense strand of a siNAmolecule.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of NucleicAcid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically-modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or a siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′, 5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing a siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the SDF-1 RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of SDF-1 gene sequences (see for example Allshire,2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297,1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al.,2002, Science, 297, 2232-2237). As such, siNA molecules of the inventioncan be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the SDF-1 RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Duplex Forming Oligonucleotides (DFO) of the Invention

In one embodiment, the invention features siNA molecules comprisingduplex forming oligonucleotides (DFO) that can self-assemble into doublestranded oligonucleotides. The duplex forming oligonucleotides of theinvention can be chemically synthesized or expressed from transcriptionunits and/or vectors. The DFO molecules of the instant invention provideuseful reagents and methods for a variety of therapeutic, diagnostic,agricultural, veterinary, target validation, genomic discovery, geneticengineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, referred toherein for convenience but not limitation as duplex formingoligonucleotides or DFO molecules, are potent mediators of sequencespecific regulation of gene expression. The oligonucleotides of theinvention are distinct from other nucleic acid sequences known in theart (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides etc.)in that they represent a class of linear polynucleotide sequences thatare designed to self-assemble into double stranded oligonucleotides,where each strand in the double stranded oligonucleotides comprises anucleotide sequence that is complementary to a target nucleic acidmolecule. Nucleic acid molecules of the invention can thus self assembleinto functional duplexes in which each strand of the duplex comprisesthe same polynucleotide sequence and each strand comprises a nucleotidesequence that is complementary to a target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assemblyof two distinct oligonucleotide sequences where the oligonucleotidesequence of one strand is complementary to the oligonucleotide sequenceof the second strand; such double stranded oligonucleotides areassembled from two separate oligonucleotides, or from a single moleculethat folds on itself to form a double stranded structure, often referredto in the field as hairpin stem-loop structure (e.g., shRNA or shorthairpin RNA). These double stranded oligonucleotides known in the artall have a common feature in that each strand of the duplex has adistinct nucleotide sequence.

Distinct from the double stranded nucleic acid molecules known in theart, the applicants have developed a novel, potentially cost effectiveand simplified method of forming a double stranded nucleic acid moleculestarting from a single stranded or linear oligonucleotide. The twostrands of the double stranded oligonucleotide formed according to theinstant invention have the same nucleotide sequence and are notcovalently linked to each other. Such double-stranded oligonucleotidesmolecules can be readily linked post-synthetically by methods andreagents known in the art and are within the scope of the invention. Inone embodiment, the single stranded oligonucleotide of the invention(the duplex forming oligonucleotide) that forms a double strandedoligonucleotide comprises a first region and a second region, where thesecond region includes a nucleotide sequence that is an inverted repeatof the nucleotide sequence in the first region, or a portion thereof,such that the single stranded oligonucleotide self assembles to form aduplex oligonucleotide in which the nucleotide sequence of one strand ofthe duplex is the same as the nucleotide sequence of the second strand.Non-limiting examples of such duplex forming oligonucleotides areillustrated in FIGS. 14 and 15. These duplex forming oligonucleotides(DFOs) can optionally include certain palindrome or repeat sequenceswhere such palindrome or repeat sequences are present in between thefirst region and the second region of the DFO.

In one embodiment, the invention features a duplex formingoligonucleotide (DFO) molecule, wherein the DFO comprises a duplexforming self complementary nucleic acid sequence that has nucleotidesequence complementary to a target nucleic acid sequence. The DFOmolecule can comprise a single self complementary sequence or a duplexresulting from assembly of such self complementary sequences.

In one embodiment, a duplex forming oligonucleotide (DFO) of theinvention comprises a first region and a second region, wherein thesecond region comprises a nucleotide sequence comprising an invertedrepeat of nucleotide sequence of the first region such that the DFOmolecule can assemble into a double stranded oligonucleotide. Suchdouble stranded oligonucleotides can act as a short interfering nucleicacid (siNA) to modulate gene expression. Each strand of the doublestranded oligonucleotide duplex formed by DFO molecules of the inventioncan comprise a nucleotide sequence region that is complementary to thesame nucleotide sequence in a target nucleic acid molecule (e.g., targetSDF-1 RNA).

In one embodiment, the invention features a single stranded DFO that canassemble into a double stranded oligonucleotide. The applicant hassurprisingly found that a single stranded oligonucleotide withnucleotide regions of self complementarity can readily assemble intoduplex oligonucleotide constructs. Such DFOs can assemble into duplexesthat can inhibit gene expression in a sequence specific manner. The DFOmolecules of the invention comprise a first region with nucleotidesequence that is complementary to the nucleotide sequence of a secondregion and where the sequence of the first region is complementary to atarget nucleic acid (e.g., RNA). The DFO can form a double strandedoligonucleotide wherein a portion of each strand of the double strandedoligonucleotide comprises a sequence complementary to a target nucleicacid sequence.

In one embodiment, the invention features a double strandedoligonucleotide, wherein the two strands of the double strandedoligonucleotide are not covalently linked to each other, and whereineach strand of the double stranded oligonucleotide comprises anucleotide sequence that is complementary to the same nucleotidesequence in a target nucleic acid molecule or a portion thereof (e.g.,SDF-1 RNA target). In another embodiment, the two strands of the doublestranded oligonucleotide share an identical nucleotide sequence of atleast about 15, preferably at least about 16, 17, 18, 19, 20, or 21nucleotides.

In one embodiment, a DFO molecule of the invention comprises a structurehaving Formula DFO-I:

5′-p-X Z X′-3′wherein Z comprises a palindromic or repeat nucleic acid sequenceoptionally with one or more modified nucleotides (e.g., nucleotide witha modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or auniversal base), for example of length about 2 to about 24 nucleotidesin even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22or 24 nucleotides), X represents a nucleic acid sequence, for example oflength of about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides),X′ comprises a nucleic acid sequence, for example of length about 1 andabout 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotidesequence complementarity to sequence X or a portion thereof, p comprisesa terminal phosphate group that can be present or absent, and whereinsequence X and Z, either independently or together, comprise nucleotidesequence that is complementary to a target nucleic acid sequence or aportion thereof and is of length sufficient to interact (e.g., basepair) with the target nucleic acid sequence or a portion thereof (e.g.,SDF-1 RNA target). For example, X independently can comprise a sequencefrom about 12 to about 21 or more (e.g., about 12, 13, 14, 15, 16, 17,18, 19, 20, 21, or more) nucleotides in length that is complementary tonucleotide sequence in a target SDF-1 RNA or a portion thereof. Inanother non-limiting example, the length of the nucleotide sequence of Xand Z together, when X is present, that is complementary to the SDF-1RNA or a portion thereof (e.g., SDF-1 RNA target) is from about 12 toabout 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or more). In yet another non-limiting example, when X isabsent, the length of the nucleotide sequence of Z that is complementaryto the target SDF-1 RNA or a portion thereof is from about 12 to about24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24, ormore). In one embodiment X, Z and X′ are independently oligonucleotides,where X and/or Z comprises a nucleotide sequence of length sufficient tointeract (e.g., base pair) with a nucleotide sequence in the SDF-1 RNAor a portion thereof (e.g., SDF-1 RNA target). In one embodiment, thelengths of oligonucleotides X and X′ are identical. In anotherembodiment, the lengths of oligonucleotides X and X′ are not identical.In another embodiment, the lengths of oligonucleotides X and Z, or Z andX′, or X, Z and X′ are either identical or different.

When a sequence is described in this specification as being of“sufficient” length to interact (i.e., base pair) with another sequence,it is meant that the the length is such that the number of bonds (e.g.,hydrogen bonds) formed between the two sequences is enough to enable thetwo sequence to form a duplex under the conditions of interest. Suchconditions can be in vitro (e.g., for diagnostic or assay purposes) orin vivo (e.g., for therapeutic purposes). It is a simple and routinematter to determine such lengths.

In one embodiment, the invention features a double strandedoligonucleotide construct having Formula DFO-I(a):

5′-p-X Z X′-3′ 3′-X′ Z X-p-5′wherein Z comprises a palindromic or repeat nucleic acid sequence orpalindromic or repeat-like nucleic acid sequence with one or moremodified nucleotides (e.g., nucleotides with a modified base, such as2-amino purine, 2-amino-1,6-dihydro purine or a universal base), forexample of length about 2 to about 24 nucleotides in even numbers (e.g.,about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides), Xrepresents a nucleic acid sequence, for example of length about 1 toabout 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X′ comprises anucleic acid sequence, for example of length about 1 to about 21nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequencecomplementarity to sequence X or a portion thereof, p comprises aterminal phosphate group that can be present or absent, and wherein eachX and Z independently comprises a nucleotide sequence that iscomplementary to a target nucleic acid sequence or a portion thereof(e.g., SDF-1 RNA target) and is of length sufficient to interact withthe target nucleic acid sequence of a portion thereof (e.g., SDF-1 RNAtarget). For example, sequence X independently can comprise a sequencefrom about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14,15, 16, 17, 18, 19, 20, 21, or more) in length that is complementary toa nucleotide sequence in a target RNA or a portion thereof (e.g., SDF-1RNA target). In another non-limiting example, the length of thenucleotide sequence of X and Z together (when X is present) that iscomplementary to the target RNA or a portion thereof is from about 12 toabout 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or more). In yet another non-limiting example, when X isabsent, the length of the nucleotide sequence of Z that is complementaryto the target RNA or a portion thereof is from about 12 to about 24 ormore nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24 or more). Inone embodiment X, Z and X′ are independently oligonucleotides, where Xand/or Z comprises a nucleotide sequence of length sufficient tointeract (e.g., base pair) with nucleotide sequence in the target RNA ora portion thereof (e.g., SDF-1 RNA target). In one embodiment, thelengths of oligonucleotides X and X′ are identical. In anotherembodiment, the lengths of oligonucleotides X and X′ are not identical.In another embodiment, the lengths of oligonucleotides X and Z or Z andX′ or X, Z and X′ are either identical or different. In one embodiment,the double stranded oligonucleotide construct of Formula I(a) includesone or more, specifically 1, 2, 3 or 4, mismatches, to the extent suchmismatches do not significantly diminish the ability of the doublestranded oligonucleotide to inhibit target gene expression.

In one embodiment, a DFO molecule of the invention comprises structurehaving Formula DFO-II:

5′-p-X X′-3′wherein each X and X′ are independently oligonucleotides of length about12 nucleotides to about 21 nucleotides, wherein X comprises, forexample, a nucleic acid sequence of length about 12 to about 21nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21nucleotides), X′ comprises a nucleic acid sequence, for example oflength about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16,17, 18, 19, 20, or 21 nucleotides) having nucleotide sequencecomplementarity to sequence X or a portion thereof, p comprises aterminal phosphate group that can be present or absent, and wherein Xcomprises a nucleotide sequence that is complementary to a targetnucleic acid sequence (e.g., SDF-1 RNA) or a portion thereof and is oflength sufficient to interact (e.g., base pair) with the target nucleicacid sequence of a portion thereof. In one embodiment, the length ofoligonucleotides X and X′ are identical. In another embodiment thelength of oligonucleotides X and X′ are not identical. In oneembodiment, length of the oligonucleotides X and X′ are sufficient toform a relatively stable double stranded oligonucleotide.

In one embodiment, the invention features a double strandedoligonucleotide construct having Formula DFO-II(a):

5′-p-X X′-3′ 3′-X′ X-p-5′wherein each X and X′ are independently oligonucleotides of length about12 nucleotides to about 21 nucleotides, wherein X comprises a nucleicacid sequence, for example of length about 12 to about 21 nucleotides(e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X′comprises a nucleic acid sequence, for example of length about 12 toabout 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or21 nucleotides) having nucleotide sequence complementarity to sequence Xor a portion thereof, p comprises a terminal phosphate group that can bepresent or absent, and wherein X comprises nucleotide sequence that iscomplementary to a target nucleic acid sequence or a portion thereof(e.g., SDF-1 RNA target) and is of length sufficient to interact (e.g.,base pair) with the target nucleic acid sequence (e.g., SDF-1 RNA) or aportion thereof. In one embodiment, the lengths of oligonucleotides Xand X′ are identical. In another embodiment, the lengths ofoligonucleotides X and X′ are not identical. In one embodiment, thelengths of the oligonucleotides X and X′ are sufficient to form arelatively stable double stranded oligonucleotide. In one embodiment,the double stranded oligonucleotide construct of Formula II(a) includesone or more, specifically 1, 2, 3 or 4 , mismatches, to the extent suchmismatches do not significantly diminish the ability of the doublestranded oligonucleotide to inhibit target gene expression.

In one embodiment, the invention features a DFO molecule having FormulaDFO-I(b):

5′-p-Z-3′where Z comprises a palindromic or repeat nucleic acid sequenceoptionally including one or more non-standard or modified nucleotides(e.g., nucleotide with a modified base, such as 2-amino purine or auniversal base) that can facilitate base-pairing with other nucleotides.Z can be, for example, of length sufficient to interact (e.g., basepair) with nucleotide sequence of a target nucleic acid (e.g., SDF-1RNA) molecule, preferably of length of at least 12 nucleotides,specifically about 12 to about 24 nucleotides (e.g., about 12, 14, 16,18, 20, 22 or 24 nucleotides). p represents a terminal phosphate groupthat can be present or absent.

In one embodiment, a DFO molecule having any of Formula DFO-I, DFO-I(a),DFO-I(b), DFO-II(a) or DFO-II can comprise chemical modifications asdescribed herein without limitation, such as, for example, nucleotideshaving any of Formulae I-VII, stabilization chemistries as described inTable IV, or any other combination of modified nucleotides andnon-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modifiednucleotide (e.g., nucleotide with a modified base, such as 2-aminopurine or a universal base) in Z of DFO constructs having Formula DFO-I,DFO-I(a) and DFO-I(b), comprises chemically modified nucleotides thatare able to interact with a portion of the target nucleic acid sequence(e.g., modified base analogs that can form Watson Crick base pairs ornon-Watson Crick base pairs).

In one embodiment, a DFO molecule of the invention, for example a DFOhaving Formula DFO-I or DFO-II, comprises about 15 to about 40nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).In one embodiment, a DFO molecule of the invention comprises one or morechemical modifications. In a non-limiting example, the introduction ofchemically modified nucleotides and/or non-nucleotides into nucleic acidmolecules of the invention provides a powerful tool in overcomingpotential limitations of in vivo stability and bioavailability inherentto unmodified RNA molecules that are delivered exogenously. For example,the use of chemically modified nucleic acid molecules can enable a lowerdose of a particular nucleic acid molecule for a given therapeuticeffect since chemically modified nucleic acid molecules tend to have alonger half-life in serum or in cells or tissues. Furthermore, certainchemical modifications can improve the bioavailability and/or potency ofnucleic acid molecules by not only enhancing half-life but alsofacilitating the targeting of nucleic acid molecules to particularorgans, cells or tissues and/or improving cellular uptake of the nucleicacid molecules. Therefore, even if the activity of a chemically modifiednucleic acid molecule is reduced in vitro as compared to anative/unmodified nucleic acid molecule, for example when compared to anunmodified RNA molecule, the overall activity of the modified nucleicacid molecule can be greater than the native or unmodified nucleic acidmolecule due to improved stability, potency, duration of effect,bioavailability and/or delivery of the molecule.

Multifunctional or Multi-Targeted siNA Molecules of the Invention

In one embodiment, the invention features siNA molecules comprisingmultifunctional short interfering nucleic acid (multifunctional siNA)molecules that modulate the expression of one or more genes in abiologic system, such as a cell, tissue, or organism. Themultifunctional short interfering nucleic acid (multifunctional siNA)molecules of the invention can target more than one region a targetnucleic acid sequence or can target sequences of more than one distincttarget nucleic acid molecules. The multifunctional siNA molecules of theinvention can be chemically synthesized or expressed from transcriptionunits and/or vectors. The multifunctional siNA molecules of the instantinvention provide useful reagents and methods for a variety of humanapplications, therapeutic, cosmetic, diagnostic, agricultural,veterinary, target validation, genomic discovery, genetic engineeringand pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, referred toherein for convenience but not limitation as multifunctional shortinterfering nucleic acid or multifunctional siNA molecules, are potentmediators of sequence specific regulation of gene expression. Themultifunctional siNA molecules of the invention are distinct from othernucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA,shRNA, antisense oligonucleotides, etc.) in that they represent a classof polynucleotide molecules that are designed such that each strand inthe multifunctional siNA construct comprises a nucleotide sequence thatis complementary to a distinct nucleic acid sequence in one or moretarget nucleic acid molecules. A single multifunctional siNA molecule(generally a double-stranded molecule) of the invention can thus targetmore than one (e.g., 2, 3, 4, 5, or more) differing target nucleic acidtarget molecules. Nucleic acid molecules of the invention can alsotarget more than one (e.g., 2, 3, 4, 5, or more) region of the sametarget nucleic acid sequence. As such multifunctional siNA molecules ofthe invention are useful in down regulating or inhibiting the expressionof one or more target nucleic acid molecules. By reducing or inhibitingexpression of more than one target nucleic acid molecule with onemultifunctional siNA construct, multifunctional siNA molecules of theinvention represent a class of potent therapeutic agents that canprovide simultaneous inhibition of multiple targets within a disease orpathogen related pathway. Such simultaneous inhibition can providesynergistic therapeutic treatment strategies without the need forseparate preclinical and clinical development efforts or complexregulatory approval process.

Use of multifunctional siNA molecules that target more then one regionof a target nucleic acid molecule (e.g., messenger RNA) is expected toprovide potent inhibition of gene expression. For example, a singlemultifunctional siNA construct of the invention can target bothconserved and variable regions of a target nucleic acid molecule, suchas a SDF-1 RNA or DNA, thereby allowing down regulation or inhibition ofdifferent splice variants encoded by a single gene, or allowing fortargeting of both coding and non-coding regions of a target nucleic acidmolecule.

Generally, double stranded oligonucleotides are formed by the assemblyof two distinct oligonucleotides where the oligonucleotide sequence ofone strand is complementary to the oligonucleotide sequence of thesecond strand; such double stranded oligonucleotides are generallyassembled from two separate oligonucleotides (e.g., siRNA). Alternately,a duplex can be formed from a single molecule that folds on itself(e.g., shRNA or short hairpin RNA). These double strandedoligonucleotides are known in the art to mediate RNA interference andall have a common feature wherein only one nucleotide sequence region(guide sequence or the antisense sequence) has complementarity to atarget nucleic acid sequence, and the other strand (sense sequence)comprises nucleotide sequence that is homologous to the target nucleicacid sequence. Generally, the antisense sequence is retained in theactive RISC and guides the RISC to the target nucleotide sequence bymeans of complementary base-pairing of the antisense sequence with thetarget sequence for mediating sequence-specific RNA interference. It isknown in the art that in some cell culture systems, certain types ofunmodified siRNAs can exhibit “off target” effects. It is hypothesizedthat this off-target effect involves the participation of the sensesequence instead of the antisense sequence of the siRNA in the RISC (seefor example Schwarz et al., 2003, Cell, 115, 199-208). In this instancethe sense sequence is believed to direct the RISC to a sequence(off-target sequence) that is distinct from the intended targetsequence, resulting in the inhibition of the off-target sequence. Inthese double stranded nucleic acid molecules, each strand iscomplementary to a distinct target nucleic acid sequence. However, theoff-targets that are affected by these dsRNAs are not entirelypredictable and are non-specific.

Distinct from the double stranded nucleic acid molecules known in theart, the applicants have developed a novel, potentially cost effectiveand simplified method of down regulating or inhibiting the expression ofmore than one target nucleic acid sequence using a singlemultifunctional siNA construct. The multifunctional siNA molecules ofthe invention are designed to be double-stranded or partially doublestranded, such that a portion of each strand or region of themultifunctional siNA is complementary to a target nucleic acid sequenceof choice. As such, the multifunctional siNA molecules of the inventionare not limited to targeting sequences that are complementary to eachother, but rather to any two differing target nucleic acid sequences.Multifunctional siNA molecules of the invention are designed such thateach strand or region of the multifunctional siNA molecule, that iscomplementary to a given target nucleic acid sequence, is of suitablelength (e.g., from about 16 to about 28 nucleotides in length,preferably from about 18 to about 28 nucleotides in length) formediating RNA interference against the target nucleic acid sequence. Thecomplementarity between the target nucleic acid sequence and a strand orregion of the multifunctional siNA must be sufficient (at least about 8base pairs) for cleavage of the target nucleic acid sequence by RNAinterference. multifunctional siNA of the invention is expected tominimize off-target effects seen with certain siRNA sequences, such asthose described in Schwarz et al., supra.

It has been reported that dsRNAs of length between 29 base pairs and 36base pairs (Tuschl et al., International PCT Publication No. WO02/44321) do not mediate RNAi. One reason these dsRNAs are inactive maybe the lack of turnover or dissociation of the strand that interactswith the target RNA sequence, such that the is not able to efficientlyinteract with multiple copies of the target RNA resulting in asignificant decrease in the potency and efficiency of the RNAi process.Applicant has surprisingly found that the multifunctional siNAs of theinvention can overcome this hurdle and are capable of enhancing theefficiency and potency of RNAi process. As such, in certain embodimentsof the invention, multifunctional siNAs of length of about 29 to about36 base pairs can be designed such that, a portion of each strand of themultifunctional siNA molecule comprises a nucleotide sequence regionthat is complementary to a target nucleic acid of length sufficient tomediate RNAi efficiently (e.g., about 15 to about 23 base pairs) and anucleotide sequence region that is not complementary to the targetnucleic acid. By having both complementary and non-complementaryportions in each strand of the multifunctional siNA, the multifunctionalsiNA can mediate RNA interference against a target nucleic acid sequencewithout being prohibitive to turnover or dissociation (e.g., where thelength of each strand is too long to mediate RNAi against the respectivetarget nucleic acid sequence). Furthermore, design of multifunctionalsiNA molecules of the invention with internal overlapping regions allowsthe multifunctional siNA molecules to be of favorable (decreased) sizefor mediating RNA interference and of size that is well suited for useas a therapeutic agent (e.g., wherein each strand is independently fromabout 18 to about 28 nucleotides in length). Non-limiting examples areillustrated in FIGS. 16-28.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a first region and a second region, where the first region ofthe multifunctional siNA comprises a nucleotide sequence complementaryto a nucleic acid sequence of a first target nucleic acid molecule, andthe second region of the multifunctional siNA comprises nucleic acidsequence complementary to a nucleic acid sequence of a second targetnucleic acid molecule. In one embodiment, a multifunctional siNAmolecule of the invention comprises a first region and a second region,where the first region of the multifunctional siNA comprises nucleotidesequence complementary to a nucleic acid sequence of the first region ofa target nucleic acid molecule, and the second region of themultifunctional siNA comprises nucleotide sequence complementary to anucleic acid sequence of a second region of a the target nucleic acidmolecule. In another embodiment, the first region and second region ofthe multifunctional siNA can comprise separate nucleic acid sequencesthat share some degree of complementarity (e.g., from about 1 to about10 complementary nucleotides). In certain embodiments, multifunctionalsiNA constructs comprising separate nucleic acid sequences can bereadily linked post-synthetically by methods and reagents known in theart and such linked constructs are within the scope of the invention.Alternately, the first region and second region of the multifunctionalsiNA can comprise a single nucleic acid sequence having some degree ofself complementarity, such as in a hairpin or stem-loop structure.Non-limiting examples of such double stranded and hairpinmultifunctional short interfering nucleic acids are illustrated in FIGS.16 and 17 respectively. These multifunctional short interfering nucleicacids (multifunctional siNAs) can optionally include certain overlappingnucleotide sequence where such overlapping nucleotide sequence ispresent in between the first region and the second region of themultifunctional siNA (see for example FIGS. 18 and 19).

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein eachstrand of the the multifunctional siNA independently comprises a firstregion of nucleic acid sequence that is complementary to a distincttarget nucleic acid sequence and the second region of nucleotidesequence that is not complementary to the target sequence. The targetnucleic acid sequence of each strand is in the same target nucleic acidmolecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to a target nucleic acid sequence (complementaryregion 1) and a region having no sequence complementarity to the targetnucleotide sequence (non-complementary region 1); (b) the second strandof the multifunction siNA comprises a region having sequencecomplementarity to a target nucleic acid sequence that is distinct fromthe target nucleotide sequence complementary to the first strandnucleotide sequence (complementary region 2), and a region having nosequence complementarity to the target nucleotide sequence ofcomplementary region 2 (non-complementary region 2); (c) thecomplementary region 1 of the first strand comprises a nucleotidesequence that is complementary to a nucleotide sequence in thenon-complementary region 2 of the second strand and the complementaryregion 2 of the second strand comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in the non-complementary region 1of the first strand. The target nucleic acid sequence of complementaryregion 1 and complementary region 2 is in the same target nucleic acidmolecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to a target nucleic acid sequence derived from a gene(complementary region 1) and a region having no sequence complementarityto the target nucleotide sequence of complementary region 1(non-complementary region 1); (b) the second strand of the multifunctionsiNA comprises a region having sequence complementarity to a targetnucleic acid sequence derived from a gene that is distinct from the geneof complementary region 1 (complementary region 2), and a region havingno sequence complementarity to the target nucleotide sequence ofcomplementary region 2 (non-complementary region 2); (c) thecomplementary region 1 of the first strand comprises a nucleotidesequence that is complementary to a nucleotide sequence in thenon-complementary region 2 of the second strand and the complementaryregion 2 of the second strand comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in the non-complementary region 1of the first strand.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to a target nucleic acid sequence derived from a firstgene (complementary region 1) and a region having no sequencecomplementarity to the target nucleotide sequence of complementaryregion 1 (non-complementary region 1); (b) the second strand of themultifunction siNA comprises a region having sequence complementarity toa second target nucleic acid sequence distinct from the first targetnucleic acid sequence of complementary region 1 (complementary region2), provided, however, that the target nucleic acid sequence forcomplementary region 1 and target nucleic acid sequence forcomplementary region 2 are both derived from the same gene, and a regionhaving no sequence complementarity to the target nucleotide sequence ofcomplementary region 2 (non-complementary region 2); (c) thecomplementary region 1 of the first strand comprises a nucleotidesequence that is complementary to a nucleotide sequence in thenon-complementary region 2 of the second strand and the complementaryregion 2 of the second strand comprises a nucleotide sequence that iscomplementary to nucleotide sequence in the non-complementary region 1of the first strand.

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein themultifunctional siNA comprises two complementary nucleic acid sequencesin which the first sequence comprises a first region having nucleotidesequence complementary to nucleotide sequence within a first targetnucleic acid molecule, and in which the second sequence comprises afirst region having nucleotide sequence complementary to a distinctnucleotide sequence within the same target nucleic acid molecule.Preferably, the first region of the first sequence is also complementaryto the nucleotide sequence of the second region of the second sequence,and where the first region of the second sequence is complementary tothe nucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein themultifunctional siNA comprises two complementary nucleic acid sequencesin which the first sequence comprises a first region having a nucleotidesequence complementary to a nucleotide sequence within a first targetnucleic acid molecule, and in which the second sequence comprises afirst region having a nucleotide sequence complementary to a distinctnucleotide sequence within a second target nucleic acid molecule.Preferably, the first region of the first sequence is also complementaryto the nucleotide sequence of the second region of the second sequence,and where the first region of the second sequence is complementary tothe nucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional siNAmolecule comprising a first region and a second region, where the firstregion comprises a nucleic acid sequence having about 18 to about 28nucleotides complementary to a nucleic acid sequence within a firsttarget nucleic acid molecule, and the second region comprises nucleotidesequence having about 18 to about 28 nucleotides complementary to adistinct nucleic acid sequence within a second target nucleic acidmolecule.

In one embodiment, the invention features a multifunctional siNAmolecule comprising a first region and a second region, where the firstregion comprises nucleic acid sequence having about 18 to about 28nucleotides complementary to a nucleic acid sequence within a targetnucleic acid molecule, and the second region comprises nucleotidesequence having about 18 to about 28 nucleotides complementary to adistinct nucleic acid sequence within the same target nucleic acidmolecule.

In one embodiment, the invention features a double strandedmultifunctional short interfering nucleic acid (multifunctional siNA)molecule, wherein one strand of the multifunctional siNA comprises afirst region having nucleotide sequence complementary to a first targetnucleic acid sequence, and the second strand comprises a first regionhaving a nucleotide sequence complementary to a second target nucleicacid sequence. The first and second target nucleic acid sequences can bepresent in separate target nucleic acid molecules or can be differentregions within the same target nucleic acid molecule. As such,multifunctional siNA molecules of the invention can be used to targetthe expression of different genes, splice variants of the same gene,both mutant and conserved regions of one or more gene transcripts, orboth coding and non-coding sequences of the same or differing genes orgene transcripts.

In one embodiment, a target nucleic acid molecule of the inventionencodes a single protein. In another embodiment, a target nucleic acidmolecule encodes more than one protein (e.g., 1, 2, 3, 4, 5 or moreproteins). As such, a multifunctional siNA construct of the inventioncan be used to down regulate or inhibit the expression of severalproteins. For example, a multifunctional siNA molecule comprising aregion in one strand having nucleotide sequence complementarity to afirst target nucleic acid sequence derived from a gene encoding oneprotein and the second strand comprising a region with nucleotidesequence complementarity to a second target nucleic acid sequencepresent in target nucleic acid molecules derived from genes encoding twoor more proteins (e.g., two or more differing target sequences) can beused to down regulate, inhibit, or shut down a particular biologicpathway by targeting, for example, two or more targets involved in abiologic pathway.

In one embodiment the invention takes advantage of conserved nucleotidesequences present in different isoforms of cytokines or ligands andreceptors for the cytokines or ligands. By designing multifunctionalsiNAs in a manner where one strand includes a sequence that iscomplementary to a target nucleic acid sequence conserved among variousisoforms of a cytokine and the other strand includes sequence that iscomplementary to a target nucleic acid sequence conserved among thereceptors for the cytokine, it is possible to selectively andeffectively modulate or inhibit a biological pathway or multiple genesin a biological pathway using a single multifunctional siNA.

In one embodiment, a double stranded multifunctional siNA molecule ofthe invention comprises a structure having Formula MF-I:

5′-p-X Z X′-3′ 3′-Y′ Z Y-p-5′wherein each 5′-p-XZX′-3′ and 5′-p-YZY′-3′ are independently anoligonucleotide of length of about 20 nucleotides to about 300nucleotides, preferably of about 20 to about 200 nucleotides, about 20to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 toabout 40 nucleotides, about 24 to about 38 nucleotides, or about 26 toabout 38 nucleotides; XZ comprises a nucleic acid sequence that iscomplementary to a first target nucleic acid sequence; YZ is anoligonucleotide comprising nucleic acid sequence that is complementaryto a second target nucleic acid sequence; Z comprises nucleotidesequence of length about 1 to about 24 nucleotides (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 nucleotides) that is self complimentary; X comprisesnucleotide sequence of length about 1 to about 100 nucleotides,preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21nucleotides) that is complementary to nucleotide sequence present inregion Y′; Y comprises nucleotide sequence of length about 1 to about100 nucleotides, preferably about 1- about 21 nucleotides (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or21 nucleotides) that is complementary to nucleotide sequence present inregion X′; each p comprises a terminal phosphate group that isindependently present or absent; each XZ and YZ is independently oflength sufficient to stably interact (i.e., base pair) with the firstand second target nucleic acid sequence, respectively, or a portionthereof. For example, each sequence X and Y can independently comprisesequence from about 12 to about 21 or more nucleotides in length (e.g.,about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that iscomplementary to a target nucleotide sequence in different targetnucleic acid molecules, such as SDF-1, vascular endothelial growthfactor (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), vascularendothelial growth factor receptor (e.g., VEGFR1, VEGFR2, and/orVEGFR3), hypoxia induced growth factor (e.g., HIF-1), Angiopoietin(e.g., ANG1, ANG2, ANG3 and/or ANG4), Endothelial Cell Growth Factor(e.g., ECGF1), placental derived growth factor (PGF), and/or complementfactor H RNAs or a portion thereof. In another non-limiting example, thelength of the nucleotide sequence of X and Z together that iscomplementary to the first target nucleic acid sequence or a portionthereof is from about 12 to about 21 or more nucleotides (e.g., about12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In anothernon-limiting example, the length of the nucleotide sequence of Y and Ztogether, that is complementary to the second target nucleic acidsequence or a portion thereof is from about 12 to about 21 or morenucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, ormore). In one embodiment, the first target nucleic acid sequence and thesecond target nucleic acid sequence are present in the same targetnucleic acid molecule (e.g., SDF-1 RNA). In another embodiment, thefirst target nucleic acid sequence and the second target nucleic acidsequence are present in different target nucleic acid molecules. In oneembodiment, Z comprises a palindrome or a repeat sequence. In oneembodiment, the lengths of oligonucleotides X and X′ are identical. Inanother embodiment, the lengths of oligonucleotides X and X′ are notidentical. In one embodiment, the lengths of oligonucleotides Y and Y′are identical. In another embodiment, the lengths of oligonucleotides Yand Y′ are not identical. In one embodiment, the double strandedoligonucleotide construct of Formula I(a) includes one or more,specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches donot significantly diminish the ability of the double strandedoligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a structure having Formula MF-II:

5′-p-X X′-3′ 3′-Y′ Y-p-5′wherein each 5′-p-XX′-3′ and 5′-p-YY′-3′ are independently anoligonucleotide of length of about 20 nucleotides to about 300nucleotides, preferably about 20 to about 200 nucleotides, about 20 toabout 100 nucleotides, about 20 to about 40 nucleotides, about 20 toabout 40 nucleotides, about 24 to about 38 nucleotides, or about 26 toabout 38 nucleotides; X comprises a nucleic acid sequence that iscomplementary to a first target nucleic acid sequence; Y is anoligonucleotide comprising nucleic acid sequence that is complementaryto a second target nucleic acid sequence; X comprises a nucleotidesequence of length about 1 to about 100 nucleotides, preferably about 1to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that iscomplementary to nucleotide sequence present in region Y′; Y comprisesnucleotide sequence of length about 1 to about 100 nucleotides,preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21nucleotides) that is complementary to nucleotide sequence present inregion X′; each p comprises a terminal phosphate group that isindependently present or absent; each X and Y independently is of lengthsufficient to stably interact (i.e., base pair) with the first andsecond target nucleic acid sequence, respectively, or a portion thereof.For example, each sequence X and Y can independently comprise sequencefrom about 12 to about 21 or more nucleotides in length (e.g., about 12,13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to atarget nucleotide sequence in different target nucleic acid molecules ora portion thereof. In one embodiment, the first target nucleic acidsequence and the second target nucleic acid sequence are present in thesame target nucleic acid molecule (e.g., SDF-1 RNA or DNA). In anotherembodiment, the first target nucleic acid sequence and the second targetnucleic acid sequence are present in different target nucleic acidmolecules, such as SDF-1, vascular endothelial growth factor (e.g.,VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), vascular endothelial growthfactor receptor (e.g., VEGFR1, VEGFR2, and/or VEGFR3), hypoxia inducedgrowth factor (e.g., HIF-1), Angiopoietin (e.g., ANG1, ANG2, ANG3 and/orANG4), Endothelial Cell Growth Factor (e.g., ECGF1), placental derivedgrowth factor (PGF), and/or complement factor H target polynucleotidesor a portion thereof. In one embodiment, Z comprises a palindrome or arepeat sequence. In one embodiment, the lengths of oligonucleotides Xand X′ are identical. In another embodiment, the lengths ofoligonucleotides X and X′ are not identical. In one embodiment, thelengths of oligonucleotides Y and Y′ are identical. In anotherembodiment, the lengths of oligonucleotides Y and Y′ are not identical.In one embodiment, the double stranded oligonucleotide construct ofFormula I(a) includes one or more, specifically 1, 2, 3 or 4,mismatches, to the extent such mismatches do not significantly diminishthe ability of the double stranded oligonucleotide to inhibit targetgene expression.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a structure having Formula MF-III:

X   X′ Y′-W-Ywherein each X, X′, Y, and Y′ is independently an oligonucleotide oflength of about 15 nucleotides to about 50 nucleotides, preferably about18 to about 40 nucleotides, or about 19 to about 23 nucleotides; Xcomprises nucleotide sequence that is complementary to nucleotidesequence present in region Y′; X′ comprises nucleotide sequence that iscomplementary to nucleotide sequence present in region Y; each X and X′is independently of length sufficient to stably interact (i.e., basepair) with a first and a second target nucleic acid sequence,respectively, or a portion thereof; W represents a nucleotide ornon-nucleotide linker that connects sequences Y′ and Y; and themultifunctional siNA directs cleavage of the first and second targetsequence via RNA interference. In one embodiment, the first targetnucleic acid sequence and the second target nucleic acid sequence arepresent in the same target nucleic acid molecule (e.g., SDF-1 RNA). Inanother embodiment, the first target nucleic acid sequence and thesecond target nucleic acid sequence are present in different targetnucleic acid molecules, such as SDF-1, vascular endothelial growthfactor (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), vascularendothelial growth factor receptor (e.g., VEGFR1, VEGFR2, and/orVEGFR3), hypoxia induced growth factor (e.g., HIF-1), Angiopoietin(e.g., ANG1, ANG2, ANG3 and/or ANG4), Endothelial Cell Growth Factor(e.g., ECGF1), placental derived growth factor (PGF), and/or complementfactor H target polynucleotides or a portion thereof. In one embodiment,region W connects the 3′-end of sequence Y′ with the 3′-end of sequenceY. In one embodiment, region W connects the 3′-end of sequence Y′ withthe 5′-end of sequence Y. In one embodiment, region W connects the5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment,region W connects the 5′-end of sequence Y′ with the 3′-end of sequenceY. In one embodiment, a terminal phosphate group is present at the5′-end of sequence X. In one embodiment, a terminal phosphate group ispresent at the 5′-end of sequence X′. In one embodiment, a terminalphosphate group is present at the 5′-end of sequence Y. In oneembodiment, a terminal phosphate group is present at the 5′-end ofsequence Y′. In one embodiment, W connects sequences Y and Y′ via abiodegradable linker. In one embodiment, W further comprises aconjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a structure having Formula MF-IV:

X   X′ Y′-W-Ywherein each X, X′, Y, and Y′ is independently an oligonucleotide oflength of about 15 nucleotides to about 50 nucleotides, preferably about18 to about 40 nucleotides, or about 19 to about 23 nucleotides; Xcomprises nucleotide sequence that is complementary to nucleotidesequence present in region Y′; X′ comprises nucleotide sequence that iscomplementary to nucleotide sequence present in region Y; each Y and Y′is independently of length sufficient to stably interact (i.e., basepair) with a first and a second target nucleic acid sequence,respectively, or a portion thereof; W represents a nucleotide ornon-nucleotide linker that connects sequences Y′ and Y; and themultifunctional siNA directs cleavage of the first and second targetsequence via RNA interference. In one embodiment, the first targetnucleic acid sequence and the second target nucleic acid sequence arepresent in the same target nucleic acid molecule (e.g., SDF-1 RNA). Inanother embodiment, the first target nucleic acid sequence and thesecond target nucleic acid sequence are present in different targetnucleic acid molecules, such as SDF-1, vascular endothelial growthfactor (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), vascularendothelial growth factor receptor (e.g., VEGFR1, VEGFR2, and/orVEGFR3), hypoxia induced growth factor (e.g., HIF-1), Angiopoietin(e.g., ANG1, ANG2, ANG3 and/or ANG4), Endothelial Cell Growth Factor(e.g., ECGF1), placental derived growth factor (PGF), and/or complementfactor H target polynucleotides or a portion thereof. In one embodiment,region W connects the 3′-end of sequence Y′ with the 3′-end of sequenceY. In one embodiment, region W connects the 3′-end of sequence Y′ withthe 5′-end of sequence Y. In one embodiment, region W connects the5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment,region W connects the 5′-end of sequence Y′ with the 3′-end of sequenceY. In one embodiment, a terminal phosphate group is present at the5′-end of sequence X. In one embodiment, a terminal phosphate group ispresent at the 5′-end of sequence X′. In one embodiment, a terminalphosphate group is present at the 5′-end of sequence Y. In oneembodiment, a terminal phosphate group is present at the 5′-end ofsequence Y′. In one embodiment, W connects sequences Y and Y′ via abiodegradable linker. In one embodiment, W further comprises aconjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a structure having Formula MF-V:

X   X′ Y′-W-Ywherein each X, X′, Y, and Y′ is independently an oligonucleotide oflength of about 15 nucleotides to about 50 nucleotides, preferably about18 to about 40 nucleotides, or about 19 to about 23 nucleotides; Xcomprises nucleotide sequence that is complementary to nucleotidesequence present in region Y′; X′ comprises nucleotide sequence that iscomplementary to nucleotide sequence present in region Y; each X, X′, Y,or Y′ is independently of length sufficient to stably interact (i.e.,base pair) with a first, second, third, or fourth target nucleic acidsequence, respectively, or a portion thereof; W represents a nucleotideor non-nucleotide linker that connects sequences Y′ and Y; and themultifunctional siNA directs cleavage of the first, second, third,and/or fourth target sequence via RNA interference. In one embodiment,the first, second, third and fourth target nucleic acid sequence are allpresent in the same target nucleic acid molecule (e.g., SDF-1 RNA). Inanother embodiment, the first, second, third and fourth target nucleicacid sequence are independently present in different target nucleic acidmolecules, such as SDF-1, vascular endothelial growth factor (e.g.,VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), vascular endothelial growthfactor receptor (e.g., VEGFR1, VEGFR2, and/or VEGFR3), hypoxia inducedgrowth factor (e.g., HIF-1), Angiopoietin (e.g., ANG1, ANG2, ANG3 and/orANG4), Endothelial Cell Growth Factor (e.g., ECGF1), placental derivedgrowth factor (PGF), and/or complement factor H target polynucleotidesor a portion thereof. In one embodiment, region W connects the 3′-end ofsequence Y′ with the 3′-end of sequence Y. In one embodiment, region Wconnects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In oneembodiment, region W connects the 5′-end of sequence Y′ with the 5′-endof sequence Y. In one embodiment, region W connects the 5′-end ofsequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminalphosphate group is present at the 5′-end of sequence X. In oneembodiment, a terminal phosphate group is present at the 5′-end ofsequence X′. In one embodiment, a terminal phosphate group is present atthe 5′-end of sequence Y. In one embodiment, a terminal phosphate groupis present at the 5′-end of sequence Y′. In one embodiment, W connectssequences Y and Y′ via a biodegradable linker. In one embodiment, Wfurther comprises a conjugate, label, aptamer, ligand, lipid, orpolymer.

In one embodiment, regions X and Y of multifunctional siNA molecule ofthe invention (e.g., having any of Formula MF-I-MF-V), are complementaryto different target nucleic acid sequences that are portions of the sametarget nucleic acid molecule. In one embodiment, such target nucleicacid sequences are at different locations within the coding region of aRNA transcript. In one embodiment, such target nucleic acid sequencescomprise coding and non-coding regions of the same RNA transcript. Inone embodiment, such target nucleic acid sequences comprise regions ofalternately spliced transcripts or precursors of such alternatelyspliced transcripts.

In one embodiment, a multifunctional siNA molecule having any of FormulaMF-I-MF-V can comprise chemical modifications as described hereinwithout limitation, such as, for example, nucleotides having any ofFormulae I-VII described herein, stabilization chemistries as describedin Table IV, or any other combination of modified nucleotides andnon-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modifiednucleotide (e.g., nucleotide with a modified base, such as 2-aminopurine or a universal base) in Z of multifunctional siNA constructshaving Formula MF-I or MF-II comprises chemically modified nucleotidesthat are able to interact with a portion of the target nucleic acidsequence (e.g., modified base analogs that can form Watson Crick basepairs or non-Watson Crick base pairs).

In one embodiment, a multifunctional siNA molecule of the invention, forexample each strand of a multifunctional siNA having MF-I-MF-V,independently comprises about 15 to about 40 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, amultifunctional siNA molecule of the invention comprises one or morechemical modifications. In a non-limiting example, the introduction ofchemically modified nucleotides and/or non-nucleotides into nucleic acidmolecules of the invention provides a powerful tool in overcomingpotential limitations of in vivo stability and bioavailability inherentto unmodified RNA molecules that are delivered exogenously. For example,the use of chemically modified nucleic acid molecules can enable a lowerdose of a particular nucleic acid molecule for a given therapeuticeffect since chemically modified nucleic acid molecules tend to have alonger half-life in serum or in cells or tissues. Furthermore, certainchemical modifications can improve the bioavailability and/or potency ofnucleic acid molecules by not only enhancing half-life but alsofacilitating the targeting of nucleic acid molecules to particularorgans, cells or tissues and/or improving cellular uptake of the nucleicacid molecules. Therefore, even if the activity of a chemically modifiednucleic acid molecule is reduced in vitro as compared to anative/unmodified nucleic acid molecule, for example when compared to anunmodified RNA molecule, the overall activity of the modified nucleicacid molecule can be greater than the native or unmodified nucleic acidmolecule due to improved stability, potency, duration of effect,bioavailability and/or delivery of the molecule.

In another embodiment, the invention features multifunctional siNAs,wherein the multifunctional siNAs are assembled from two separatedouble-stranded siNAs, with one of the ends of each sense strand istethered to the end of the sense strand of the other siNA molecule, suchthat the two antisense siNA strands are annealed to their correspondingsense strand that are tethered to each other at one end (see FIG. 22).The tethers or linkers can be nucleotide-based linkers or non-nucleotidebased linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 5′-end of one sense strand of the siNAis tethered to the 5′-end of the sense strand of the other siNAmolecule, such that the 5′-ends of the two antisense siNA strands,annealed to their corresponding sense strand that are tethered to eachother at one end, point away (in the opposite direction) from each other(see FIG. 22 (A)). The tethers or linkers can be nucleotide-basedlinkers or non-nucleotide based linkers as generally known in the artand as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 3′-end of one sense strand of the siNAis tethered to the 3′-end of the sense strand of the other siNAmolecule, such that the 5′-ends of the two antisense siNA strands,annealed to their corresponding sense strand that are tethered to eachother at one end, face each other (see FIG. 22(B)). The tethers orlinkers can be nucleotide-based linkers or non-nucleotide based linkersas generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 5′-end of one sense strand of the siNAis tethered to the 3′-end of the sense strand of the other siNAmolecule, such that the 5′-end of the one of the antisense siNA strandsannealed to their corresponding sense strand that are tethered to eachother at one end, faces the 3′-end of the other antisense strand (seeFIGS. 22(C-D)). The tethers or linkers can be nucleotide-based linkersor non-nucleotide based linkers as generally known in the art and asdescribed herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 5′-end of one antisense strand of thesiNA is tethered to the 3′-end of the antisense strand of the other siNAmolecule, such that the 5′-end of the one of the sense siNA strandsannealed to their corresponding antisense sense strand that are tetheredto each other at one end, faces the 3′-end of the other sense strand(see FIGS. 22(G-H)). In one embodiment, the linkage between the 5′-endof the first antisense strand and the 3′-end of the second antisensestrand is designed in such a way as to be readily cleavable (e.g.,biodegradable linker) such that the 5′end of each antisense strand ofthe multifunctional siNA has a free 5′-end suitable to mediate RNAinterefence-based cleavage of the target RNA. The tethers or linkers canbe nucleotide-based linkers or non-nucleotide based linkers as generallyknown in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 5′-end of one antisense strand of thesiNA is tethered to the 5′-end of the antisense strand of the other siNAmolecule, such that the 3′-end of the one of the sense siNA strandsannealed to their corresponding antisense sense strand that are tetheredto each other at one end, faces the 3′-end of the other sense strand(see FIG. 22(E)). In one embodiment, the linkage between the 5′-end ofthe first antisense strand and the 5′-end of the second antisense strandis designed in such a way as to be readily cleavable (e.g.,biodegradable linker) such that the 5′end of each antisense strand ofthe multifunctional siNA has a free 5′-end suitable to mediate RNAinterefence-based cleavage of the target RNA. The tethers or linkers canbe nucleotide-based linkers or non-nucleotide based linkers as generallyknown in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 3′-end of one antisense strand of thesiNA is tethered to the 3′-end of the antisense strand of the other siNAmolecule, such that the 5′-end of the one of the sense siNA strandsannealed to their corresponding antisense sense strand that are tetheredto each other at one end, faces the 3′-end of the other sense strand(see FIG. 22(F)). In one embodiment, the linkage between the 5′-end ofthe first antisense strand and the 5′-end of the second antisense strandis designed in such a way as to be readily cleavable (e.g.,biodegradable linker) such that the 5′end of each antisense strand ofthe multifunctional siNA has a free 5′-end suitable to mediate RNAinterefence-based cleavage of the target RNA. The tethers or linkers canbe nucleotide-based linkers or non-nucleotide based linkers as generallyknown in the art and as described herein.

In any of the above embodiments, a first target nucleic acid sequence orsecond target nucleic acid sequence can independently comprise SDF-1RNA, DNA or a portion thereof. In one embodiment, the first targetnucleic acid sequence is a SDF-1 RNA, DNA or a portion thereof and thesecond target nucleic acid sequence is a SDF-1 RNA, DNA of a portionthereof. In one embodiment, the first target nucleic acid sequence is aSDF-1 RNA, DNA or a portion thereof and the second target nucleic acidsequence is a vascular endothelial growth factor (e.g., VEGF-A, VEGF-B,VEGF-C, and/or VEGF-D), vascular endothelial growth factor receptor(e.g., VEGFR1, VEGFR2, and/or VEGFR3), hypoxia induced growth factor(e.g., HIF-1), Angiopoietin (e.g., ANG1, ANG2, ANG3 and/or ANG4),Endothelial Cell Growth Factor (e.g., ECGF1), placental derived growthfactor (PGF), and/or complement factor H target RNA, DNA of a portionthereof.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table III outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table III outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA·3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA·3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe SDF-1 RNA has been modulated long enough to reduce the levels of theundesirable protein. This period of time varies between hours to daysdepending upon the disease state. Improvements in the chemical synthesisof RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677;Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated byreference herein)) have expanded the ability to modify nucleic acidmolecules by introducing nucleotide modifications to enhance theirnuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′, 4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule of the invention or thesense and antisense strands of a siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-o-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more5′ and/or a 3′-cap structure, for example, on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of cap moieties areshown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′, 5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to an —C(O)—NH—R, where R is eitheralkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′,where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to prevent ortreat diseases, traits, disorders, and/or conditions described herein orotherwise known in the art to be related to SDF-1 gene expression,and/or any other trait, disease, disorder or condition that is relatedto or will respond to the levels of SDF-1 polynucleotides or proteinsexpressed therefrom in a cell or tissue, alone or in combination withother therapies.

In one embodiment, a siNA composition of the invention can comprise adelivery vehicle, including liposomes, for administration to a subject,carriers and diluents and their salts, and/or can be present inpharmaceutically acceptable formulations (for non-limiting examples ofdelivery vehicles and formulations, see for example U.S. Ser. No.60/678531, filed May 6, 2005, incorporated by reference herein). Methodsfor the delivery of nucleic acid molecules are described in Akhtar etal., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for AntisenseOligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999,Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp.Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752,184-192, all of which are incorporated herein by reference. Beigelman etal., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595further describe the general methods for delivery of nucleic acidmolecules. These protocols can be utilized for the delivery of virtuallyany nucleic acid molecule. Nucleic acid molecules can be administered tocells by a variety of methods known to those of skill in the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such asbiodegradable polymers, hydrogels, cyclodextrins (see for exampleGonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al.,International PCT publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactos amine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in United StatesPatent Application Publication No. 20030077829, incorporated byreference herein in its entirety.

In one embodiment, a siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed withdelivery systems as described in U.S. Patent Application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In one embodiment, siNA molecules and compositions of the invention forthe treatment of ocular conditions (e.g., macular degeneration, diabeticretinopathy etc.) are administered to a subject intraocularly or byintraocular means. In another embodiment, siNA molecules andcompositions of the invention for the treatment of ocular conditions(e.g., macular degeneration, diabetic retinopathy etc.) are administeredto a subject periocularly or by periocular means (see for exampleAhlheim et al., International PCT publication No. WO 03/24420). In oneembodiment, a siNA molecule, composition and/or formulation of theinvention is administered to a subject intraocularly or by intraocularmeans. In another embodiment, a siNA molecule, composition and/orformulation of the invention is administered to a subject periocularlyor by periocular means. Periocular administration generally provides aless invasive approach to administering siNA molecules and formualtionor composition thereof to a subject (see for example Ahlheim et al.,International PCT publication No. WO 03/24420). The use of periocularadministraction also minimizes the risk of retinal detachment, allowsfor more frequent dosing or administraction, provides a clinicallyrelevant route of administraction for macular degeneration, diabeticretinopathy and other optic conditions, and also provides thepossiblilty of using resevoirs (e.g., implants, pumps or other devices)for drug delivery. In one embodiment, siNA compounds and compositions ofthe invention are administered locally, e.g., via intraocular orperiocular means, such as injection, iontophoresis (see, for example, WO03/043689 and WO 03/030989), contact lens, or implant, about every 1-50weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 weeks), alone or in combination with other compounds and/or therapeisherein. In one embodiment, siNA compounds and compositions of theinvention are administered systemically (e.g., via intravenous,subcutaneous, intramuscular, infusion, pump, implant etc.) about every1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 weeks), alone or in combination with other compounds and/ortherapies described herein and/or otherwise known in the art.

In one embodiment, a siNA molecule of the invention is administerediontophoretically, for example to a particular organ or compartment(e.g., the eye, back of the eye, heart, liver, kidney, bladder,prostate, tumor, CNS etc.). Non-limiting examples of iontophoreticdelivery are described in, for example, WO 03/043689 and WO 03/030989,which are incorporated by reference in their entireties herein.

In one embodiment, the nucleic acid molecules of the invention areadministered via pulmonary delivery, such as by inhalation of an aerosolor spray dried formulation administered by an inhalation device ornebulizer, providing rapid local uptake of the nucleic acid moleculesinto relevant pulmonary tissues. Solid particulate compositionscontaining respirable dry particles of micronized nucleic acidcompositions can be prepared by grinding dried or lyophilized nucleicacid compositions, and then passing the micronized composition through,for example, a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprising the nucleicacid compositions of the invention can optionally contain a dispersantwhich serves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition ofthe invention can be produced by any suitable means, such as with anebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers comprise the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, for example, sodium chloride or other suitable salts.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxybenzoate, anti-oxidants,flavorings, volatile oils, buffering agents and emulsifiers and otherformulation surfactants. The aerosols of solid particles comprising theactive composition and surfactant can likewise be produced with anysolid particulate aerosol generator. Aerosol generators foradministering solid particulate therapeutics to a subject produceparticles which are respirable, as explained above, and generate avolume of aerosol containing a predetermined metered dose of atherapeutic composition at a rate suitable for human administration.

In one embodiment, a solid particulate aerosol generator of theinvention is an insufflator. Suitable formulations for administration byinsufflation include finely comminuted powders which can be delivered bymeans of an insufflator. In the insufflator, the powder, e.g., a metereddose thereof effective to carry out the treatments described herein, iscontained in capsules or cartridges, typically made of gelatin orplastic, which are either pierced or opened in situ and the powderdelivered by air drawn through the device upon inhalation or by means ofa manually-operated pump. The powder employed in the insufflatorconsists either solely of the active ingredient or of a powder blendcomprising the active ingredient, a suitable powder diluent, such aslactose, and an optional surfactant. The active ingredient typicallycomprises from 0.1 to 100 w/w of the formulation. A second type ofillustrative aerosol generator comprises a metered dose inhaler. Metereddose inhalers are pressurized aerosol dispensers, typically containing asuspension or solution formulation of the active ingredient in aliquified propellant. During use these devices discharge the formulationthrough a valve adapted to deliver a metered volume to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, for example US PatentApplication No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728;6,565,885, all incorporated by reference herein.

In one embodiment, the invention features the use of methods to deliverthe nucleic acid molecules of the instant invention to the centralnervous system and/or peripheral nervous system. Experiments havedemonstrated the efficient in vivo uptake of nucleic acids by neurons.As an example of local administration of nucleic acids to nerve cells,Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe astudy in which a 15 mer phosphorothioate antisense nucleic acid moleculeto c-fos is administered to rats via microinjection into the brain.Antisense molecules labeled with tetramethylrhodamine-isothiocyanate(TRITC) or fluorescein isothiocyanate (FITC) were taken up byexclusively by neurons thirty minutes post-injection. A diffusecytoplasmic staining and nuclear staining was observed in these cells.As an example of systemic administration of nucleic acid to nerve cells,Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an invivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotideconjugates were used to target the p75 neurotrophin receptor inneuronally differentiated PC12 cells. Following a two week course of IPadministration, pronounced uptake of p75 neurotrophin receptor antisensewas observed in dorsal root ganglion (DRG) cells. In addition, a markedand consistent down-regulation of p75 was observed in DRG neurons.Additional approaches to the targeting of nucleic acid to neurons aredescribed in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle etal., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, BrainResearch, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199;Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, BrainRes. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39.Nucleic acid molecules of the invention are therefore amenable todelivery to and uptake by cells that express repeat expansion allelicvariants for modulation of RE gene expression. The delivery of nucleicacid molecules of the invention, targeting RE is provided by a varietyof different strategies. Traditional approaches to CNS delivery that canbe used include, but are not limited to, intrathecal andintracerebroventricular administration, implantation of catheters andpumps, direct injection or perfusion at the site of injury or lesion,injection into the brain arterial system, or by chemical or osmoticopening of the blood-brain barrier. Other approaches can include the useof various transport and carrier systems, for example though the use ofconjugates and biodegradable polymers. Furthermore, gene therapyapproaches, for example as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

The delivery of nucleic acid molecules of the invention to the CNS isprovided by a variety of different strategies. Traditional approaches toCNS delivery that can be used include, but are not limited to,intrathecal and intracerebroventricular administration, implantation ofcatheters and pumps, direct injection or perfusion at the site of injuryor lesion, injection into the brain arterial system, or by chemical orosmotic opening of the blood-brain barrier. Other approaches can includethe use of various transport and carrier systems, for example though theuse of conjugates and biodegradable polymers. Furthermore, gene therapyapproaches, for example as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered to the liver as is generallyknown in the art (see for example Wen et al., 2004, World JGastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14;Liu et al., 2003, Gene Ther., 10, 180-7; Hong et al., 2003, J PharmPharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7;and Matsuno et al., 2003, Gene Ther., 10, 1559-66).

In one embodiment, the invention features the use of methods to deliverthe nucleic acid molecules of the instant invention to hematopoieticcells, including monocytes and lymphocytes. These methods are describedin detail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2),920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion andPhillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, NucleicAcids Research, 22(22), 4681-8. Such methods, as described above,include the use of free oligonucleitide, cationic lipid formulations,liposome formulations including pH sensitive liposomes andimmunoliposomes, and bioconjugates including oligonucleotides conjugatedto fusogenic peptides, for the transfection of hematopoietic cells witholigonucleotides.

In one embodiment, the siNA molecules and compositions of the inventionare administered to the inner ear by contacting the siNA with inner earcells, tissues, or structures such as the cochlea, under conditionssuitable for the administration. In one embodiment, the administrationcomprises methods and devices as described in U.S. Pat. Nos. 5,421,818,5,476,446, 5,474,529, 6,045,528, 6,440,102, 6,685,697, 6,120,484; and5,572,594; all incorporated by reference herein and the teachings ofSilverstein, 1999, Ear Nose Throat J., 78, 595-8, 600; and Jackson andSilverstein, 2002, Otolaryngol Clin North Am., 35, 639-53, and adaptedfor use the siNA molecules of the invention.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered directly or topically (e.g.,locally) to the dermis or follicles as is generally known in the art(see for example Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnieret al., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs,16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; andPreat and Dujardin, 2001, STP PharmaSciences, 11, 57-68). In oneembodiment, the siNA molecules of the invention and formulations orcompositions thereof are administered directly or topically using ahydroalcoholic gel formulation comprising an alcohol (e.g., ethanol orisopropanol), water, and optionally including additional agents suchisopropyl myristate and carbomer 980.

In one embodiment, delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) CellFectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, delivery systems of the invention include patches,tablets, suppositories, pessaries, gels and creams, and can containexcipients such as solubilizers and enhancers (e.g., propylene glycol,bile salts and amino acids), and other vehicles (e.g., polyethyleneglycol, fatty acid esters and derivatives, and hydrophilic polymers suchas hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, a siNA molecule of the invention is administerediontophoretically, for example to the dermis or to other relevanttissues such as the inner ear/cochlea. Non-limiting examples ofiontophoretic delivery are described in, for example, WO 03/043689 andWO 03/030989, which are incorporated by reference in their entiretiesherein.

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA Pharm Sci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PhramaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886;U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No.5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as creams, gels, sprays, oilsand other suitable compositions for topical, dermal, or transdermaladministration as is known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to asubject by systemic administration in a pharmaceutically acceptablecomposition or formulation. By “systemic administration” is meant invivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes that lead to systemic absorption include, without limitation:intravenous, subcutaneous, portal vein, intraperitoneal, inhalation,oral, intrapulmonary and intramuscular. Each of these administrationroutes exposes the siNA molecules of the invention to an accessiblediseased tissue. The rate of entry of a drug into the circulation hasbeen shown to be a function of molecular weight or size. The use of aliposome or other drug carrier comprising the compounds of the instantinvention can potentially localize the drug, for example, in certaintissue types, such as the tissues of the reticular endothelial system(RES). A liposome formulation that can facilitate the association ofdrug with the surface of cells, such as, lymphocytes and macrophages isalso useful. This approach can provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the nucleic acid molecules ofthe instant invention in the physical location most suitable for theirdesired activity. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: P-glycoprotein inhibitors (such as Pluronic P85); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery (Emerich, D F et al, 1999, Cell Transplant,8, 47-58); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of a composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes) andnucleic acid molecules of the invention. These formulations offer amethod for increasing the accumulation of drugs (e.g., siNA) in targettissues. This class of drug carriers resists opsonization andelimination by the mononuclear phagocytic system (MPS or RES), therebyenabling longer blood circulation times and enhanced tissue exposure forthe encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627;Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomeshave been shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,24864-24870; Choi et al., International PCT Publication No. WO 96/10391;Ansell et al., International PCT Publication No. WO 96/10390; Holland etal., International PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono-or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pat.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intra-muscular administration, by administration totarget cells ex-planted from a subject followed by reintroduction intothe subject, or by any other means that would allow for introductioninto the desired target cell (for a review see Couture et al., 1996,TIG., 12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of asiNA duplex, or a single self-complementary strand that self hybridizesinto a siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi:10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of a siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of a siNA molecule, wherein the sequence isoperably linked to the 3′-end of the open reading frame and wherein thesequence is operably linked to the initiation region, the intron, theopen reading frame and the termination region in a manner which allowsexpression and/or delivery of the siNA molecule.

SDF-1 Biology and Biochemistry

Diabetic retinoplasty is the major cause of blindness among Americansunder the age of 65. Diabetic retinoplasty is caused by oxygenstarvation in the retina, which induces aberrant neovascularizationresulting in newly formed blood vessels intruding into the vitreous ofthe eye. The new blood vessels destroy normal retinal architecture andmay hemorrhage, causing bleeding into the eye, which ultimately impairsvision.

Recent studies have shown that vitreal stromal cell-derived factor-1(SDF-1) plays a major role in proliferative retinoplasty and may be anideal target for the prevention of proliferative diabetic retinoplasty.Butler et al., 2005, J. Clin. Invest., 115, 86-93. SDF-1 is thepredominant chemokine that mobilizes hemopoietic stem cells (HSCs) andendothelial progenitor cells (EPCs). Hattori et al., 2003, Leuk.Lymphoma, 44:575-582. SDF-1 expression is is induced by a wide varietyof cell types in response to stimuli such as stress and injury. Forexample, SDF-1 has also been shown to be upregulated in many damagedtissues as part of the injury response and is thought to recruitstem/progenitor cells to the damaged tissue to promote repair. Hatch etal., 2002, Cloning Stem Cells, 4:339-352.

Recently, Butler et al., showed that SDF-1 levels in vitreous samples ofhuman patients increase with severity of proliferative diabeticretinoplasty, i.e., as the disease progresses. Patients with theseverest form of the disease were found to have a level of SDF-1 atleast 50-fold greater than the level found in normal eyes. Butler et al.further demonstrated that SDF-1 plays an important role in the migrationof HSC-derived EPCs to the site of vascular injury by regulatingmolecules involved in the injury/repair response. Specifically, theyshowed that SDF-1 induces human retinal endothelial cells to increaseexpression of VCAM-1 and reduce tight cellular junctions by reducingoccludin expression, both of which changes serve to recruit hemopoeiticand endothelial progenitor cells along an SDF-1 concentration gradient.

Using a murine model of proliferative adult retinoplasty, it has beenshown that the majority of new vessels formed in response to oxygenstarvation originate from hemopoietic stem cell-derived endothelialprogenitor cells. Grant et al., 2002, Nat. Med., 8:607-612. The murinemodel described in Grant et al. requires the administration of growthfactor (recombinant adeno-associated virus-VEGF (rAAV-VEGF)) to thevitreous of the eye and ischemic injury. Butler et al. has recentlyfurther shown that SDF-1 (at levels found in human patients withproliferative retinoplasty) induces retinoplasty in a similar murinemodel, wherein the administration of rAAV-VEGF was replaced with theadministration of recombinant SDF-1 protein within the vitreous. Weeklyinjections were performed up to 4 weeks after laser injury to maintainthe concentration of SDF-1 in the vitreous. Administration of exogenousSDF-1 was able to enhance HSC-derived EPC migration and incorporationinto the sites of ischemic injury, resulting in retinoplasty.

Butler et al. further describes the prevention of retinalneovascularization in the SDF-1 murine model using antibodies that blockSDF-1. SDF-1-specific blocking antibodies were injected into the mousevitreous at the time of laser injury. Weekly booster injections of SDF-1blocking antibody were given intravitrealy during the ischemic repairphase. Control animals received either no intravitreal injections orweekly intravitreal mock antibody injections. In contrast to controlanimals, mice treated with SDF-1 blocking antibody produced almost noHSC-derived blood vessels in response to VEGF bolus and ischemia injury.Cross-sectional histological analysis of SDF-1 blocking antibody-treatedeyes versus nontreated control eyes was also performed. The control eyesexhibited severe preretinal vascularization, as shown by the grossdisruption of the retinal architecture, in response to VEGFadministration and retinal ischemia. In contrast, none of the anti-SDF-1treated eyes exhibited retinal neovascularization, and all retained aretinal architecture similar to that of a normal retina. These resultsshowed that treating the eye with intravitreal injections of SDF-1blocking antibodies prevents retinal neovascularization.

As discussed above, the involvement of SDF-1 in the development andmaintenance of proliferative retinoplasty has been demonstrated. The useof small interfering nucleic acid molecules targeting SDF-1 provides aclass of novel therapeutic agents that can be used in the the treatmentof proliferative diabetic retinoplasty and any other disease orcondition that responds to modulation of SDF-1 genes.

Examples

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example, using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H20 followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease, trait, or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target (e.g., SDF-1) RNA sequence.These parameters include but are not limited to secondary or tertiaryRNA structure, the nucleotide base composition of the target sequence,the degree of homology between various regions of the target sequence,or the relative position of the target sequence within the RNAtranscript. Based on these determinations, any number of target siteswithin the RNA transcript can be chosen to screen siNA molecules forefficacy, for example by using in vitro RNA cleavage assays, cellculture, or animal models. In a non-limiting example, anywhere from 1 to1000 target sites are chosen within the transcript based on the size ofthe siNA construct to be used. High throughput screening assays can bedeveloped for screening siNA molecules using methods known in the art,such as with multi-well or multi-plate assays to determine efficientreduction in target (e.g., SDF-1) gene expression.

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target(e.g., SDF-1) gene but are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Table II). If terminalTT residues are desired for the sequence (as described in paragraph 7),then the two 3′ terminal nucleotides of both the sense and antisensestrands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the SDF-1 RNA sequence.

10. Other design considerations can be used when selecting targetnucleic acid sequences, see, for example, Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004,doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32,doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a targetsequence is used to screen for target sites in cells expressing target(e.g., SDF-1) RNA, such as cultured Jurkat, HeLa, A549 or 293T cells.The general strategy used in this approach is shown in FIG. 9. Cellsexpressing the target (e.g., SDF-1) RNA are transfected with the pool ofsiNA constructs and cells that demonstrate a phenotype associated withtarget inhibition are sorted. The pool of siNA constructs can beexpressed from transcription cassettes inserted into appropriate vectors(see for example FIG. 7 and FIG. 8). The siNA from cells demonstrating apositive phenotypic change (e.g., decreased proliferation, decreasedtarget mRNA levels or decreased target protein expression), aresequenced to determine the most suitable target site(s) within thetarget (e.g., SDF-1) RNA sequence.

Example 4 siNA Design

siNA target sites were chosen by analyzing sequences of the target(e.g., SDF-1) RNA target and optionally prioritizing the target sites onthe basis of folding (structure of any given sequence analyzed todetermine siNA accessibility to the target), by using a library of siNAmolecules as described in Example 3, or alternately by using an in vitrosiNA system as described in Example 6 herein. siNA molecules weredesigned that could bind each target and are optionally individuallyanalyzed by computer folding to assess whether the siNA molecule caninteract with the target sequence. Varying the length of the siNAmolecules can be chosen to optimize activity. Generally, a sufficientnumber of complementary nucleotide bases are chosen to bind to, orotherwise interact with, the target (e.g., SDF-1) RNA, but the degree ofcomplementarity can be modulated to accommodate siNA duplexes or varyinglength or base composition. By using such methodologies, siNA moleculescan be designed to target sites within any known RNA sequence, forexample those RNA sequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat.No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringesupra, incorporated by reference herein in their entireties.Additionally, deprotection conditions can be modified to provide thebest possible yield and purity of siNA constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting target (e.g., SDF-1) RNA targets.The assay comprises the system described by Tuschl et al., 1999, Genesand Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with a SDF-1 RNA. A Drosophila extract derived fromsyncytial blastoderm is used to reconstitute RNAi activity in vitro.Target RNA is generated via in vitro transcription from an appropriatetarget expressing plasmid using T7 RNA polymerase or via chemicalsynthesis as described herein. Sense and antisense siNA strands (forexample 20 uM each) are annealed by incubation in buffer (such as 100 mMpotassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysisbuffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate). Annealing can be monitored by gelelectrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophila lysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10%[vol/vol] lysis buffer containing siNA (10 nM final concentration). Thereaction mixture also contains 10 mM creatine phosphate, 10 ug/mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25× Passive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich siNA is omitted from the reaction.

Alternately, internally-labeled target (e.g., SDF-1) RNA for the assayis prepared by in vitro transcription in the presence of [alpha-³²P]CTP, passed over a G50 Sephadex column by spin chromatography and usedas SDF-1 RNA without further purification. Optionally, SDF-1 RNA is5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays areperformed as described above and target (e.g., SDF-1) RNA and thespecific RNA cleavage products generated by RNAi are visualized on anautoradiograph of a gel. The percentage of cleavage is determined byPHOSPHOR IMAGER® (autoradiography) quantitation of bands representingintact control RNA or RNA from control reactions without siNA and thecleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites in thetarget (e.g., SDF-1) RNA target for siNA mediated RNAi cleavage, whereina plurality of siNA constructs are screened for RNAi mediated cleavageof the target (e.g., SDF-1) RNA target, for example, by analyzing theassay reaction by electrophoresis of labeled target (e.g., SDF-1) RNA,or by northern blotting, as well as by other methodology well known inthe art.

Example 7 Nucleic Acid Inhibition of Target (e.g., SDF-1) RNA In Vivo

siNA molecules targeted to the human target (e.g., SDF-1) RNA aredesigned and synthesized as described above. These nucleic acidmolecules can be tested for cleavage activity in vivo, for example,using the following procedure.

Two formats are used to test the efficacy of siNAs against a giventarget. First, the reagents are tested in cell culture using, forexample, Jurkat, HeLa, A549 or 293T cells, to determine the extent ofRNA and protein inhibition. siNA reagents are selected against thetarget as described herein. RNA inhibition is measured after delivery ofthese reagents by a suitable transfection agent to, for example, Jurkat,HeLa, A549 or 293T cells. Relative amounts of target (e.g., SDF-1) RNAare measured versus actin using real-time PCR monitoring ofamplification (eg., ABI 7700 TAQMAN®). A comparison is made to a mixtureof oligonucleotide sequences made to unrelated targets or to arandomized siNA control with the same overall length and chemistry, butrandomly substituted at each position. Primary and secondary leadreagents are chosen for the target and optimization performed. After anoptimal transfection agent concentration is chosen, a RNA time-course ofinhibition is performed with the lead siNA molecule. In addition, acell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., Jurkat, HeLa, A549 or 293T cells) are seeded, for example,at 1×10⁵ cells per well of a six-well dish in EGM-2 (BioWhittaker) theday before transfection. siNA (final concentration, for example 20 nM)and cationic lipid (e.g., final concentration 2 μg/ml) are complexed inEGM basal media (Biowhittaker) at 37° C. for 30 minutes in polystyrenetubes. Following vortexing, the complexed siNA is added to each well andincubated for the times indicated. For initial optimization experiments,cells are seeded, for example, at 1×10³ in 96 well plates and siNAcomplex added as described. Efficiency of delivery of siNA to cells isdetermined using a fluorescent siNA complexed with lipid. Cells in6-well dishes are incubated with siNA for 24 hours, rinsed with PBS andfixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptakeof siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/reaction) and normalizing to β-actin or GAPDH mRNAin parallel TAQMAN® reactions (real-time PCR monitoring ofamplification). For each gene of interest an upper and lower primer anda fluorescently labeled probe are designed. Real time incorporation ofSYBR Green I dye into a specific PCR product can be measured in glasscapillary tubes using a lightcyler. A standard curve is generated foreach primer pair using control cRNA. Values are represented as relativeexpression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Models Useful to Evaluate the Down-Regulation of SDF-1 GeneExpression

Evaluating the efficacy of siNA molecules of the invention in animalmodels is an important prerequisite to human clinical trials. Variousanimal models of cancer, proliferative, ocular, respiratory, etc.diseases, conditions, or disorders as are known in the art can beadapted for use for pre-clinical evaluation of the efficacy of nucleicacid compositions of the invetention in modulating target (e.g., SDF-1)gene expression toward therapeutic or research use. In a non-limitingexample, an animal model of proliferative retinopathy as described inButler et al., 2005, J. Clin, Invest., 115, 86-93, is utilized toevaluate the efficacy of siNA molecules and compositions of theinvention.

Example 9 RNAi Mediated Inhibition of SDF-1 Gene Expression

In Vitro siNA Mediated Inhibition of SDF-1 RNA

siNA constructs (are tested for efficacy in reducing SDF-1 RNAexpression in cells, (e.g., HEKn/HEKa, HeLa, A549, A375 cells). Cellsare plated approximately 24 hours before transfection in 96-well platesat 5,000-7,500 cells/well, 100 μl/well, such that at the time oftransfection cells are 70-90% confluent. For transfection, annealedsiNAs are mixed with the transfection reagent (Lipofectamine 2000,Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes atroom temperature. The siNA transfection mixtures are added to cells togive a final siNA concentration of 25 nM in a volume of 150 μl. EachsiNA transfection mixture is added to 3 wells for triplicate siNAtreatments. Cells are incubated at 37° for 24 hours in the continuedpresence of the siNA transfection mixture. At 24 hours, RNA is preparedfrom each well of treated cells. The supernatants with the transfectionmixtures are first removed and discarded, then the cells are lysed andRNA prepared from each well. Target gene expression following treatmentis evaluated by RT-PCR for the SDF-1 gene and for a control gene (36B4,an RNA polymerase subunit) for normalization. The triplicate data isaveraged and the standard deviations determined for each treatment.Normalized data are graphed and the percent reduction of target mRNA byactive siNAs in comparison to their respective inverted control siNAs isdetermined.

Example 10 Indications

Particular conditions and disease states that can be associated withgene expression modulation include, but are not limited to cancer,proliferative, ocular, respiratory, kidney etc. diseases, conditions, ordisorders as described herein or otherwise known in the art, and anyother diseases, conditions or disorders that are related to or willrespond to the levels of a target (e.g., target SDF-1 protein or targetSDF-1 polynucleotide) in a cell or tissue, alone or in combination withother therapies.

Example 11 Multifunctional siNA Inhibition of SDF-1 RNA Expression

Multifunctional siNA Design

Once target sites have been identified for multifunctional siNAconstructs, each strand of the siNA is designed with a complementaryregion of length, for example, of about 18 to about 28 nucleotides, thatis complementary to a different target nucleic acid sequence. Eachcomplementary region is designed with an adjacent flanking region ofabout 4 to about 22 nucleotides that is not complementary to the targetsequence, but which comprises complementarity to the complementaryregion of the other sequence (see for example FIG. 16). Hairpinconstructs can likewise be designed (see for example FIG. 17).Identification of complementary, palindrome or repeat sequences that areshared between the different target nucleic acid sequences can be usedto shorten the overall length of the multifunctional siNA constructs(see for example FIGS. 18 and 19).

In a non-limiting example, three additional categories of additionalmultifunctional siNA designs are presented that allow a single siNAmolecule to silence multiple targets. The first method utilizes linkersto join siNAs (or multiunctional siNAs) in a direct manner. This canallow the most potent siNAs to be joined without creating a long,continuous stretch of RNA that has potential to trigger an interferonresponse. The second method is a dendrimeric extension of theoverlapping or the linked multifunctional design; or alternatively theorganization of siNA in a supramolecular format. The third method useshelix lengths greater than 30 base pairs. Processing of these siNAs byDicer will reveal new, active 5′ antisense ends. Therefore, the longsiNAs can target the sites defined by the original 5′ ends and thosedefined by the new ends that are created by Dicer processing. When usedin combination with traditional multifunctional siNAs (where the senseand antisense strands each define a target) the approach can be used forexample to target 4 or more sites.

I. Tethered Bifunctional siNAs

The basic idea is a novel approach to the design of multifunctionalsiNAs in which two antisense siNA strands are annealed to a single sensestrand. The sense strand oligonucleotide contains a linker (e.g.,non-nulcoetide linker as described herein) and two segments that annealto the antisense siNA strands (see FIG. 22). The linkers can alsooptionally comprise nucleotide-based linkers. Several potentialadvantages and variations to this approach include, but are not limitedto:

-   1. The two antisense siNAs are independent. Therefore, the choice of    target sites is not constrained by a requirement for sequence    conservation between two sites. Any two highly active siNAs can be    combined to form a multifunctional siNA.-   2. When used in combination with target sites having homology, siNAs    that target a sequence present in two genes (e.g., different    isoforms), the design can be used to target more than two sites. A    single multifunctional siNA can be for example, used to SDF-1 RNA of    two different SDF-1 RNAs.-   3. Multifunctional siNAs that use both the sense and antisense    strands to target a gene can also be incorporated into a tethered    multifuctional design. This leaves open the possibility of targeting    6 or more sites with a single complex.-   4. It can be possible to anneal more than two antisense strand siNAs    to a single tethered sense strand.-   5. The design avoids long continuous stretches of dsRNA. Therefore,    it is less likely to initiate an interferon response.-   6. The linker (or modifications attached to it, such as conjugates    described herein) can improve the pharmacokinetic properties of the    complex or improve its incorporation into liposomes. Modifications    introduced to the linker should not impact siNA activity to the same    extent that they would if directly attached to the siNA (see for    example FIGS. 27 and 28).-   7. The sense strand can extend beyond the annealed antisense strands    to provide additional sites for the attachment of conjugates.-   8. The polarity of the complex can be switched such that both of the    antisense 3′ ends are adjacent to the linker and the 5′ ends are    distal to the linker or combination thereof.    Dendrimer and Supramolecular siNAs

In the dendrimer siNA approach, the synthesis of siNA is initiated byfirst synthesizing the dendrimer template followed by attaching variousfunctional siNAs. Various constructs are depicted in FIG. 23. The numberof functional siNAs that can be attached is only limited by thedimensions of the dendrimer used.

Supramolecular Approach to Multifunctional siNA

The supramolecular format simplifies the challenges of dendrimersynthesis. In this format, the siNA strands are synthesized by standardRNA chemistry, followed by annealing of various complementary strands.The individual strand synthesis contains an antisense sense sequence ofone siNA at the 5′-end followed by a nucleic acid or synthetic linker,such as hexaethyleneglyol, which in turn is followed by sense strand ofanother siNA in 5′ to 3′ direction. Thus, the synthesis of siNA strandscan be carried out in a standard 3′ to 5′ direction. Representativeexamples of trifunctional and tetrafunctional siNAs are depicted in FIG.24. Based on a similar principle, higher functionality siNA constructscan be designed as long as efficient annealing of various strands isachieved.

Dicer Enabled Multifunctional siNA

Using bioinformatic analysis of multiple targets, stretches of identicalsequences shared between differing target sequences can be identifiedranging from about two to about fourteen nucleotides in length. Theseidentical regions can be designed into extended siNA helixes (e.g., >30base pairs) such that the processing by Dicer reveals a secondaryfunctional 5′-antisense site (see for example FIG. 25). For example,when the first 17 nucleotides of a siNA antisense strand (e.g., 21nucleotide strands in a duplex with 3′-TT overhangs) are complementaryto a SDF-1 RNA, robust silencing was observed at 25 nM. 80% silencingwas observed with only 16 nucleotide complementarity in the same format.

Incorporation of this property into the designs of siNAs of about 30 to40 or more base pairs results in additional multifunctional siNAconstructs. The example in FIG. 25 illustrates how a 30 base-pair duplexcan target three distinct sequences after processing by Dicer-RNaseIII;these sequences can be on the same mRNA or separate RNAs, such as viraland host factor messages, or multiple points along a given pathway(e.g., inflammatory cascades). Furthermore, a 40 base-pair duplex cancombine a bifunctional design in tandem, to provide a single duplextargeting four target sequences. An even more extensive approach caninclude use of homologous sequences to enable five or six targetssilenced for one multifunctional duplex. The example in FIG. 25demonstrates how this can be achieved. A 30 base pair duplex is cleavedby Dicer into 22 and 8 base pair products from either end (8 b.p.fragments not shown). For ease of presentation the overhangs generatedby dicer are not shown—but can be compensated for. Three targetingsequences are shown. The required sequence identity overlapped isindicated by grey boxes. The N's of the parent 30 b.p. siNA aresuggested sites of 2′-OH positions to enable Dicer cleavage if this istested in stabilized chemistries. Note that processing of a 30 merduplex by Dicer RNase III does not give a precise 22+8 cleavage, butrather produces a series of closely related products (with 22+8 beingthe primary site). Therefore, processing by Dicer will yield a series ofactive siNAs. Another non-limiting example is shown in FIG. 26. A 40base pair duplex is cleaved by Dicer into 20 base pair products fromeither end. For ease of presentation the overhangs generated by dicerare not shown—but can be compensated for. Four targeting sequences areshown in four colors, blue, light-blue and red and orange. The requiredsequence identity overlapped is indicated by grey boxes. This designformat can be extended to larger RNAs. If chemically stabilized siNAsare bound by Dicer, then strategically located ribonucleotide linkagescan enable designer cleavage products that permit our more extensiverepertoire of multifunctional designs. For example cleavage products notlimited to the Dicer standard of approximately 22-nucleotides can allowmultifunctional siNA constructs with a target sequence identity overlapranging from, for example, about 3 to about 15 nucleotides.

Example 12 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the SDF-1 RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the SDF-1 RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of SDF-1 RNAs with siNAmolecules can be used to inhibit gene expression and define the role ofspecified gene products in the progression of disease or infection. Inthis manner, other genetic targets can be defined as important mediatorsof the disease. These experiments will lead to better treatment of thedisease progression by affording the possibility of combinationtherapies (e.g., multiple siNA molecules targeted to different genes,siNA molecules coupled with known small molecule inhibitors, orintermittent treatment with combinations siNA molecules and/or otherchemical or biological molecules). Other in vitro uses of siNA moleculesof this invention are well known in the art, and include detection ofthe presence of mRNAs associated with a disease, infection, or relatedcondition. Such RNA is detected by determining the presence of acleavage product after treatment with a siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the SDF-1 RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of SDF-1 RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of SDF-1 RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments, optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I SDF-1 Accession Numbers 1: NM_199168 Homo sapiens chemokine(C-X-C motif) ligand 12 (stromal cell- derived factor 1) (CXCL12), mRNAgi|40316923|ref|NM_199168.1|[40316923] 2: U19495 Human intercrine-alpha(hIRH) mRNA, complete cds gi|1754834|gb|U19495.1|HSU19495[1754834] 3:BC039893 Homo sapiens chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1), mRNA (cDNA clone MGC: 47612 IMAGE: 5729604), completecds gi|25058963|gb|BC039893.1|[25058963] 4: L36034 Human pre-B cellstimulating factor homologue (SDF1a) mRNA, complete cdsgi|1220363|gb|L36034.1|HUMSDF1A[1220363] 5: AY802782 Homo sapienschemokine (C-X-C motif) ligand 12 (stromal cell- derived factor 1)(CXCL12) gene, complete cds gi|55375972|gb|AY802782.1|[55375972] 6:AY874118 Homo sapiens stromal cell-derived factor 1a mRNA, complete cdsgi|58760242|gb|AY874118.1|[58760242] 7: NM_000609 Homo sapiens chemokine(C-X-C motif) ligand 12 (stromal cell- derived factor 1) (CXCL12), mRNAgi|40316922|ref|NM_000609.3|[40316922] 8: L36033 Human pre-B cellstimulating factor homologue (SDF1b) mRNA, complete cdsgi|1220365|gb|L36033.1|HUMSDF1B[1220365] 9: U16752 Human cytokineSDF-1-beta mRNA, complete cds gi|1272194|gb|U16752.1|HSU16752[1272194]10: AY644456 Homo sapiens stromal cell-derived factor 1 gamma (CXCL12)mRNA, complete cds gi|50400179|gb|AY644456.1|[50400179]

TABLE II SDF-1 siNA and Target Sequences Seq Seq Seq Pos Seq ID UPosUpper seq ID LPos Lower seq ID CXCL12a NM_199168.1 3 CGCACUUUCACUCUCCGUC1 3 CGCACUUUCACUCUCCGUC 1 21 GACGGAGAGUGAAAGUGCG 109 21CAGCCGCAUUGCCCGCUCG 2 21 CAGCCGCAUUGCCCGCUCG 2 39 CGAGCGGGCAAUGCGGCUG110 39 GGCGUCCGGCCCCCGACCC 3 39 GGCGUCCGGCCCCCGACCC 3 57GGGUCGGGGGCCGGACGCC 111 57 CGCGCUCGUCCGCCCGCCC 4 57 CGCGCUCGUCCGCCCGCCC4 75 GGGCGGGCGGACGAGCGCG 112 75 CGCCCGCCCGCCCGCGCCA 5 75CGCCCGCCCGCCCGCGCCA 5 93 UGGCGCGGGCGGGCGGGCG 113 93 AUGAACGCCAAGGUCGUGG6 93 AUGAACGCCAAGGUCGUGG 6 111 CCACGACCUUGGCGUUCAU 114 111GUCGUGCUGGUCCUCGUGC 7 111 GUCGUGCUGGUCCUCGUGC 7 129 GCACGAGGACCAGCACGAC115 129 CUGACCGCGCUCUGCCUCA 8 129 CUGACCGCGCUCUGCCUCA 8 147UGAGGCAGAGCGCGGUCAG 116 147 AGCGACGGGAAGCCCGUCA 9 147AGCGACGGGAAGCCCGUCA 9 165 UGACGGGCUUCCCGUCGCU 117 165AGCCUGAGCUACAGAUGCC 10 165 AGCCUGAGCUACAGAUGCC 10 183GGCAUCUGUAGCUCAGGCU 118 183 CCAUGCCGAUUCUUCGAAA 11 183CCAUGCCGAUUCUUCGAAA 11 201 UUUCGAAGAAUCGGCAUGG 119 201AGCCAUGUUGCCAGAGCCA 12 201 AGCCAUGUUGCCAGAGCCA 12 219UGGCUCUGGCAACAUGGCU 120 219 AACGUCAAGCAUCUCAAAA 13 219AACGUCAAGCAUCUCAAAA 13 237 UUUUGAGAUGCUUGACGUU 121 237AUUCUCAACACUCCAAACU 14 237 AUUCUCAACACUCCAAACU 14 255AGUUUGGAGUGUUGAGAAU 122 255 UGUGCCCUUCAGAUUGUAG 15 255UGUGCCCUUCAGAUUGUAG 15 273 CUACAAUCUGAAGGGCACA 123 273GCCCGGCUGAAGAACAACA 16 273 GCCCGGCUGAAGAACAACA 16 291UGUUGUUCUUCAGCCGGGC 124 291 AACAGACAAGUGUGCAUUG 17 291AACAGACAAGUGUGCAUUG 17 309 CAAUGCACACUUGUCUGUU 125 309GACCCGAAGCUAAAGUGGA 18 309 GACCCGAAGCUAAAGUGGA 18 327UCCACUUUAGCUUCGGGUC 126 327 AUUCAGGAGUACCUGGAGA 19 327AUUCAGGAGUACCUGGAGA 19 345 UCUCCAGGUACUCCUGAAU 127 345AAAGCUUUAAACAAGUAAG 20 345 AAAGCUUUAAACAAGUAAG 20 363CUUACUUGUUUAAAGCUUU 128 363 GCACAACAGCCAAAAAGGA 21 363GCACAACAGCCAAAAAGGA 21 381 UCCUUUUUGGCUGUUGUGC 129 381ACUUUCCGCUAGACCCACU 22 381 ACUUUCCGCUAGACCCACU 22 399AGUGGGUCUAGCGGAAAGU 130 399 UCGAGGAAAACUAAAACCU 23 399UCGAGGAAAACUAAAACCU 23 417 AGGUUUUAGUUUUCCUCGA 131 417UUGUGAGAGAUGAAAGGGC 24 417 UUGUGAGAGAUGAAAGGGC 24 435GCCCUUUCAUCUCUCACAA 132 435 CAAAGACGUGGGGGAGGGG 25 435CAAAGACGUGGGGGAGGGG 25 453 CCCCUCCCCCACGUCUUUG 133 453GGCCUUAACCAUGAGGACC 26 453 GGCCUUAACCAUGAGGACC 26 471GGUCCUCAUGGUUAAGGCC 134 471 CAGGUGUGUGUGUGGGGUG 27 471CAGGUGUGUGUGUGGGGUG 27 489 CACCCCACACACACACCUG 135 489GGGCACAUUGAUCUGGGAU 28 489 GGGCACAUUGAUCUGGGAU 28 507AUCCCAGAUCAAUGUGCCC 136 507 UCGGGCCUGAGGUUUGCCA 29 507UCGGGCCUGAGGUUUGCCA 29 525 UGGCAAACCUCAGGCCCGA 137 525AGCAUUUAGACCCUGCAUU 30 525 AGCAUUUAGACCCUGCAUU 30 543AAUGCAGGGUCUAAAUGCU 138 543 UUAUAGCAUACGGUAUGAU 31 543UUAUAGCAUACGGUAUGAU 31 561 AUCAUACCGUAUGCUAUAA 139 561UAUUGCAGCUUAUAUUCAU 32 561 UAUUGCAGCUUAUAUUCAU 32 579AUGAAUAUAAGCUGCAAUA 140 579 UCCAUGCCCUGUACCUGUG 33 579UCCAUGCCCUGUACCUGUG 33 597 CACAGGUACAGGGCAUGGA 141 597GCACGUUGGAACUUUUAUU 34 597 GCACGUUGGAACUUUUAUU 34 615AAUAAAAGUUCCAACGUGC 142 615 UACUGGGGUUUUUCUAAGA 35 615UACUGGGGUUUUUCUAAGA 35 633 UCUUAGAAAAACCCCAGUA 143 633AAAGAAAUUGUAUUAUCAA 36 633 AAAGAAAUUGUAUUAUCAA 36 651UUGAUAAUACAAUUUCUUU 144 651 ACAGCAUUUUCAAGCAGUU 37 651ACAGCAUUUUCAAGCAGUU 37 669 AACUGCUUGAAAAUGCUGU 145 669UAGUUCCUUCAUGAUCAUC 38 669 UAGUUCCUUCAUGAUCAUC 38 687GAUGAUCAUGAAGGAACUA 146 687 CACAAUCAUCAUCAUUCUC 39 687CACAAUCAUCAUCAUUCUC 39 705 GAGAAUGAUGAUGAUUGUG 147 705CAUUCUCAUUUUUUAAAUC 40 705 CAUUCUCAUUUUUUAAAUC 40 723GAUUUAAAAAAUGAGAAUG 148 723 CAACGAGUACUUCAAGAUC 41 723CAACGAGUACUUCAAGAUC 41 741 GAUCUUGAAGUACUCGUUG 149 741CUGAAUUUGGCUUGUUUGG 42 741 CUGAAUUUGGCUUGUUUGG 42 759CCAAACAAGCCAAAUUCAG 150 759 GAGCAUCUCCUCUGCUCCC 43 759GAGCAUCUCCUCUGCUCCC 43 777 GGGAGCAGAGGAGAUGCUC 151 777CCUGGGGAGUCUGGGCACA 44 777 CCUGGGGAGUCUGGGCACA 44 795UGUGCCCAGACUCCCCAGG 152 795 AGUCAGGUGGUGGCUUAAC 45 795AGUCAGGUGGUGGCUUAAC 45 813 GUUAAGCCACCACCUGACU 153 813CAGGGAGCUGGAAAAAGUG 46 813 CAGGGAGCUGGAAAAAGUG 46 831CACUUUUUCCAGCUCCCUG 154 831 GUCCUUUCUUCAGACACUG 47 831GUCCUUUCUUCAGACACUG 47 849 CAGUGUCUGAAGAAAGGAC 155 849GAGGCUCCCGCAGCAGCGC 48 849 GAGGCUCCCGCAGCAGCGC 48 867GCGCUGCUGCGGGAGCCUC 156 867 CCCCUCCCAAGAGGAAGGC 49 867CCCCUCCCAAGAGGAAGGC 49 885 GCCUUCCUCUUGGGAGGGG 157 885CCUCUGUGGCACUCAGAUA 50 885 CCUCUGUGGCACUCAGAUA 50 903UAUCUGAGUGCCACAGAGG 158 903 ACCGACUGGGGCUGGGCGC 51 903ACCGACUGGGGCUGGGCGC 51 921 GCGCCCAGCCCCAGUCGGU 159 921CCGCCACUGCCUUCACCUC 52 921 CCGCCACUGCCUUCACCUC 52 939GAGGUGAAGGCAGUGGCGG 160 939 CCUCUUUCAACCUCAGUGA 53 939CCUCUUUCAACCUCAGUGA 53 957 UCACUGAGGUUGAAAGAGG 161 957AUUGGCUCUGUGGGCUCCA 54 957 AUUGGCUCUGUGGGCUCCA 54 975UGGAGCCCACAGAGCCAAU 162 975 AUGUAGAAGCCACUAUUAC 55 975AUGUAGAAGCCACUAUUAC 55 993 GUAAUAGUGGCUUCUACAU 163 993CUGGGACUGUGCUCAGAGA 56 993 CUGGGACUGUGCUCAGAGA 56 1011UCUCUGAGCACAGUCCCAG 164 1011 ACCCCUCUCCCAGCUAUUC 57 1011ACCCCUCUCCCAGCUAUUC 57 1029 GAAUAGCUGGGAGAGGGGU 165 1029CCUACUCUCUCCCCGACUC 58 1029 CCUACUCUCUCCCCGACUC 58 1047GAGUCGGGGAGAGAGUAGG 166 1047 CCGAGAGCAUGCUUAAUCU 59 1047CCGAGAGCAUGCUUAAUCU 59 1065 AGAUUAAGCAUGCUCUCGG 167 1065UUGCUUCUGCUUCUCAUUU 60 1065 UUGCUUCUGCUUCUCAUUU 60 1083AAAUGAGAAGCAGAAGCAA 168 1083 UCUGUAGCCUGAUCAGCGC 61 1083UCUGUAGCCUGAUCAGCGC 61 1101 GCGCUGAUCAGGCUACAGA 169 1101CCGCACCAGCCGGGAAGAG 62 1101 CCGCACCAGCCGGGAAGAG 62 1119CUCUUCCCGGCUGGUGCGG 170 1119 GGGUGAUUGCUGGGGCUCG 63 1119GGGUGAUUGCUGGGGCUCG 63 1137 CGAGCCCCAGCAAUCACCC 171 1137GUGCCCUGCAUCCCUCUCC 64 1137 GUGCCCUGCAUCCCUCUCC 64 1155GGAGAGGGAUGCAGGGCAC 172 1155 CUCCCAGGGCCUGCCCCAC 65 1155CUCCCAGGGCCUGCCCCAC 65 1173 GUGGGGCAGGCCCUGGGAG 173 1173CAGCUCGGGCCCUCUGUGA 66 1173 CAGCUCGGGCCCUCUGUGA 66 1191UCACAGAGGGCCCGAGCUG 174 1191 AGAUCCGUCUUUGGCCUCC 67 1191AGAUCCGUCUUUGGCCUCC 67 1209 GGAGGCCAAAGACGGAUCU 175 1209CUCCAGAAUGGAGCUGGCC 68 1209 CUCCAGAAUGGAGCUGGCC 68 1227GGCCAGCUCCAUUCUGGAG 176 1227 CCUCUCCUGGGGAUGUGUA 69 1227CCUCUCCUGGGGAUGUGUA 69 1245 UACACAUCCCCAGGAGAGG 177 1245AAUGGUCCCCCUGCUUACC 70 1245 AAUGGUCCCCCUGCUUACC 70 1263GGUAAGCAGGGGGACCAUU 178 1263 CCGCAAAAGACAAGUCUUU 71 1263CCGCAAAAGACAAGUCUUU 71 1281 AAAGACUUGUCUUUUGCGG 179 1281UACAGAAUCAAAUGCAAUU 72 1281 UACAGAAUCAAAUGCAAUU 72 1299AAUUGCAUUUGAUUCUGUA 180 1299 UUUAAAUCUGAGAGCUCGC 73 1299UUUAAAUCUGAGAGCUCGC 73 1317 GCGAGCUCUCAGAUUUAAA 181 1317CUUUGAGUGACUGGGUUUU 74 1317 CUUUGAGUGACUGGGUUUU 74 1335AAAACCCAGUCACUCAAAG 182 1335 UGUGAUUGCCUCUGAAGCC 75 1335UGUGAUUGCCUCUGAAGCC 75 1353 GGCUUCAGAGGCAAUCACA 183 1353CUAUGUAUGCCAUGGAGGC 76 1353 CUAUGUAUGCCAUGGAGGC 76 1371GCCUCCAUGGCAUACAUAG 184 1371 CACUAACAAACUCUGAGGU 77 1371CACUAACAAACUCUGAGGU 77 1389 ACCUCAGAGUUUGUUAGUG 185 1389UUUCCGAAAUCAGAAGCGA 78 1389 UUUCCGAAAUCAGAAGCGA 78 1407UCGCUUCUGAUUUCGGAAA 186 1407 AAAAAAUCAGUGAAUAAAC 79 1407AAAAAAUCAGUGAAUAAAC 79 1425 GUUUAUUCACUGAUUUUUU 187 1425CCAUCAUCUUGCCACUACC 80 1425 CCAUCAUCUUGCCACUACC 80 1443GGUAGUGGCAAGAUGAUGG 188 1443 CCCCUCCUGAAGCCACAGC 81 1443CCCCUCCUGAAGCCACAGC 81 1461 GCUGUGGCUUCAGGAGGGG 189 1461CAGGGUUUCAGGUUCCAAU 82 1461 CAGGGUUUCAGGUUCCAAU 82 1479AUUGGAACCUGAAACCCUG 190 1479 UCAGAACUGUUGGCAAGGU 83 1479UCAGAACUGUUGGCAAGGU 83 1497 ACCUUGCCAACAGUUCUGA 191 1497UGACAUUUCCAUGCAUAAA 84 1497 UGACAUUUCCAUGCAUAAA 84 1515UUUAUGCAUGGAAAUGUCA 192 1515 AUGCGAUCCACAGAAGGUC 85 1515AUGCGAUCCACAGAAGGUC 85 1533 GACCUUCUGUGGAUCGCAU 193 1533CCUGGUGGUAUUUGUAACU 86 1533 CCUGGUGGUAUUUGUAACU 86 1551AGUUACAAAUACCACCAGG 194 1551 UUUUUGCAAGGCAUUUUUU 87 1551UUUUUGCAAGGCAUUUUUU 87 1569 AAAAAAUGCCUUGCAAAAA 195 1569UUAUAUAUAUUUUUGUGCA 88 1569 UUAUAUAUAUUUUUGUGCA 88 1587UGCACAAAAAUAUAUAUAA 196 1587 ACAUUUUUUUUUACGUUUC 89 1587ACAUUUUUUUUUACGUUUC 89 1605 GAAACGUAAAAAAAAAUGU 197 1605CUUUAGAAAACAAAUGUAU 90 1605 CUUUAGAAAACAAAUGUAU 90 1623AUACAUUUGUUUUCUAAAG 198 1623 UUUCAAAAUAUAUUUAUAG 91 1623UUUCAAAAUAUAUUUAUAG 91 1641 CUAUAAAUAUAUUUUGAAA 199 1641GUCGAACAAUUCAUAUAUU 92 1641 GUCGAACAAUUCAUAUAUU 92 1659AAUAUAUGAAUUGUUCGAC 200 1659 UUGAAGUGGAGCCAUAUGA 93 1659UUGAAGUGGAGCCAUAUGA 93 1677 UCAUAUGGCUCCACUUCAA 201 1677AAUGUCAGUAGUUUAUACU 94 1677 AAUGUCAGUAGUUUAUACU 94 1695AGUAUAAACUACUGACAUU 202 1695 UUCUCUAUUAUCUCAAACU 95 1695UUCUCUAUUAUCUCAAACU 95 1713 AGUUUGAGAUAAUAGAGAA 203 1713UACUGGCAAUUUGUAAAGA 96 1713 UACUGGCAAUUUGUAAAGA 96 1731UCUUUACAAAUUGCCAGUA 204 1731 AAAUAUAUAUGAUAUAUAA 97 1731AAAUAUAUAUGAUAUAUAA 97 1749 UUAUAUAUCAUAUAUAUUU 205 1749AAUGUGAUUGCAGCUUUUC 98 1749 AAUGUGAUUGCAGCUUUUC 98 1767GAAAAGCUGCAAUCACAUU 206 1767 CAAUGUUAGCCACAGUGUA 99 1767CAAUGUUAGCCACAGUGUA 99 1785 UACACUGUGGCUAACAUUG 207 1785AUUUUUUCACUUGUACUAA 100 1785 AUUUUUUCACUUGUACUAA 100 1803UUAGUACAAGUGAAAAAAU 208 1803 AAAUUGUAUCAAAUGUGAC 101 1803AAAUUGUAUCAAAUGUGAC 101 1821 GUCACAUUUGAUACAAUUU 209 1821CAUUAUAUGCACUAGCAAU 102 1821 CAUUAUAUGCACUAGCAAU 102 1839AUUGCUAGUGCAUAUAAUG 210 1839 UAAAAUGCUAAUUGUUUCA 103 1839UAAAAUGCUAAUUGUUUCA 103 1857 UGAAACAAUUAGCAUUUUA 211 1857AUGGUAUAAACGUCCUACU 104 1857 AUGGUAUAAACGUCCUACU 104 1875AGUAGGACGUUUAUACCAU 212 1875 UGUAUGUGGGAAUUUAUUU 105 1875UGUAUGUGGGAAUUUAUUU 105 1893 AAAUAAAUUCCCACAUACA 213 1893UACCUGAAAUAAAAUUCAU 106 1893 UACCUGAAAUAAAAUUCAU 106 1911AUGAAUUUUAUUUCAGGUA 214 1911 UUAGUUGUUAGUGAUGGAG 107 1911UUAGUUGUUAGUGAUGGAG 107 1929 CUCCAUCACUAACAACUAA 215 1920AGUGAUGGAGCUUAAAAAA 108 1920 AGUGAUGGAGCUUAAAAAA 108 1938UUUUUUAAGCUCCAUCACU 216 CXCL12b NM_000609.3 3 CGCACUUUCACUCUCCGUC 1 3CGCACUUUCACUCUCCGUC 1 21 GACGGAGAGUGAAAGUGCG 109 21 CAGCCGCAUUGCCCGCUCG2 21 CAGCCGCAUUGCCCGCUCG 2 39 CGAGCGGGCAAUGCGGCUG 110 39GGCGUCCGGCCCCCGACCC 3 39 GGCGUCCGGCCCCCGACCC 3 57 GGGUCGGGGGCCGGACGCC111 57 CGCGCUCGUCCGCCCGCCC 4 57 CGCGCUCGUCCGCCCGCCC 4 75GGGCGGGCGGACGAGCGCG 112 75 CGCCCGCCCGCCCGCGCCA 5 75 CGCCCGCCCGCCCGCGCCA5 93 UGGCGCGGGCGGGCGGGCG 113 93 AUGAACGCCAAGGUCGUGG 6 93AUGAACGCCAAGGUCGUGG 6 111 CCACGACCUUGGCGUUCAU 114 111GUCGUGCUGGUCCUCGUGC 7 111 GUCGUGCUGGUCCUCGUGC 7 129 GCACGAGGACCAGCACGAC115 129 CUGACCGCGCUCUGCCUCA 8 129 CUGACCGCGCUCUGCCUCA 8 147UGAGGCAGAGCGCGGUCAG 116 147 AGCGACGGGAAGCCCGUCA 9 147AGCGACGGGAAGCCCGUCA 9 165 UGACGGGCUUCCCGUCGCU 117 165AGCCUGAGCUACAGAUGCC 10 165 AGCCUGAGCUACAGAUGCC 10 183GGCAUCUGUAGCUCAGGCU 118 183 CCAUGCCGAUUCUUCGAAA 11 183CCAUGCCGAUUCUUCGAAA 11 201 UUUCGAAGAAUCGGCAUGG 119 201AGCCAUGUUGCCAGAGCCA 12 201 AGCCAUGUUGCCAGAGCCA 12 219UGGCUCUGGCAACAUGGCU 120 219 AACGUCAAGCAUCUCAAAA 13 219AACGUCAAGCAUCUCAAAA 13 237 UUUUGAGAUGCUUGACGUU 121 237AUUCUCAACACUCCAAACU 14 237 AUUCUCAACACUCCAAACU 14 255AGUUUGGAGUGUUGAGAAU 122 255 UGUGCCCUUCAGAUUGUAG 15 255UGUGCCCUUCAGAUUGUAG 15 273 CUACAAUCUGAAGGGCACA 123 273GCCCGGCUGAAGAACAACA 16 273 GCCCGGCUGAAGAACAACA 16 291UGUUGUUCUUCAGCCGGGC 124 291 AACAGACAAGUGUGCAUUG 17 291AACAGACAAGUGUGCAUUG 17 309 CAAUGCACACUUGUCUGUU 125 309GACCCGAAGCUAAAGUGGA 18 309 GACCCGAAGCUAAAGUGGA 18 327UCCACUUUAGCUUCGGGUC 126 327 AUUCAGGAGUACCUGGAGA 19 327AUUCAGGAGUACCUGGAGA 19 345 UCUCCAGGUACUCCUGAAU 127 345AAAGCUUUAAACAAGAGGU 217 345 AAAGCUUUAAACAAGAGGU 217 363ACCUCUUGUUUAAAGCUUU 396 363 UUCAAGAUGUGAGAGGGUC 218 363UUCAAGAUGUGAGAGGGUC 218 381 GACCCUCUCACAUCUUGAA 397 381CAGACGCCUGAGGAACCCU 219 381 CAGACGCCUGAGGAACCCU 219 399AGGGUUCCUCAGGCGUCUG 398 399 UUACAGUAGGAGCCCAGCU 220 399UUACAGUAGGAGCCCAGCU 220 417 AGCUGGGCUCCUACUGUAA 399 417UCUGAAACCAGUGUUAGGG 221 417 UCUGAAACCAGUGUUAGGG 221 435CCCUAACACUGGUUUCAGA 400 435 GAAGGGCCUGCCACAGCCU 222 435GAAGGGCCUGCCACAGCCU 222 453 AGGCUGUGGCAGGCCCUUC 401 453UCCCCUGCCAGGGCAGGGC 223 453 UCCCCUGCCAGGGCAGGGC 223 471GCCCUGCCCUGGCAGGGGA 402 471 CCCCAGGCAUUGCCAAGGG 224 471CCCCAGGCAUUGCCAAGGG 224 489 CCCUUGGCAAUGCCUGGGG 403 489GCUUUGUUUUGCACACUUU 225 489 GCUUUGUUUUGCACACUUU 225 507AAAGUGUGCAAAACAAAGC 404 507 UGCCAUAUUUUCACCAUUU 226 507UGCCAUAUUUUCACCAUUU 226 525 AAAUGGUGAAAAUAUGGCA 405 525UGAUUAUGUAGCAAAAUAC 227 525 UGAUUAUGUAGCAAAAUAC 227 543GUAUUUUGCUACAUAAUCA 406 543 CAUGACAUUUAUUUUUCAU 228 543CAUGACAUUUAUUUUUCAU 228 561 AUGAAAAAUAAAUGUCAUG 407 561UUUAGUUUGAUUAUUCAGU 229 561 UUUAGUUUGAUUAUUCAGU 229 579ACUGAAUAAUCAAACUAAA 408 579 UGUCACUGGCGACACGUAG 230 579UGUCACUGGCGACACGUAG 230 597 CUACGUGUCGCCAGUGACA 409 597GCAGCUUAGACUAAGGCCA 231 597 GCAGCUUAGACUAAGGCCA 231 615UGGCCUUAGUCUAAGCUGC 410 615 AUUAUUGUACUUGCCUUAU 232 615AUUAUUGUACUUGCCUUAU 232 633 AUAAGGCAAGUACAAUAAU 411 633UUAGAGUGUCUUUCCACGG 233 633 UUAGAGUGUCUUUCCACGG 233 651CCGUGGAAAGACACUCUAA 412 651 GAGCCACUCCUCUGACUCA 234 651GAGCCACUCCUCUGACUCA 234 669 UGAGUCAGAGGAGUGGCUC 413 669AGGGCUCCUGGGUUUUGUA 235 669 AGGGCUCCUGGGUUUUGUA 235 687UACAAAACCCAGGAGCCCU 414 687 AUUCUCUGAGCUGUGCAGG 236 687AUUCUCUGAGCUGUGCAGG 236 705 CCUGCACAGCUCAGAGAAU 415 705GUGGGGAGACUGGGCUGAG 237 705 GUGGGGAGACUGGGCUGAG 237 723CUCAGCCCAGUCUCCCCAC 416 723 GGGAGCCUGGCCCCAUGGU 238 723GGGAGCCUGGCCCCAUGGU 238 741 ACCAUGGGGCCAGGCUCCC 417 741UCAGCCCUAGGGUGGAGAG 239 741 UCAGCCCUAGGGUGGAGAG 239 759CUCUCCACCCUAGGGCUGA 418 759 GCCACCAAGAGGGACGCCU 240 759GCCACCAAGAGGGACGCCU 240 777 AGGCGUCCCUCUUGGUGGC 419 777UGGGGGUGCCAGGACCAGU 241 777 UGGGGGUGCCAGGACCAGU 241 795ACUGGUCCUGGCACCCCCA 420 795 UCAACCUGGGCAAAGCCUA 242 795UCAACCUGGGCAAAGCCUA 242 813 UAGGCUUUGCCCAGGUUGA 421 813AGUGAAGGCUUCUCUCUGU 243 813 AGUGAAGGCUUCUCUCUGU 243 831ACAGAGAGAAGCCUUCACU 422 831 UGGGAUGGGAUGGUGGAGG 244 831UGGGAUGGGAUGGUGGAGG 244 849 CCUCCACCAUCCCAUCCCA 423 849GGCCACAUGGGAGGCUCAC 245 849 GGCCACAUGGGAGGCUCAC 245 867GUGAGCCUCCCAUGUGGCC 424 867 CCCCCUUCUCCAUCCACAU 246 867CCCCCUUCUCCAUCCACAU 246 885 AUGUGGAUGGAGAAGGGGG 425 885UGGGAGCCGGGUCUGCCUC 247 885 UGGGAGCCGGGUCUGCCUC 247 903GAGGCAGACCCGGCUCCCA 426 903 CUUCUGGGAGGGCAGCAGG 248 903CUUCUGGGAGGGCAGCAGG 248 921 CCUGCUGCCCUCCCAGAAG 427 921GGCUACCCUGAGCUGAGGC 249 921 GGCUACCCUGAGCUGAGGC 249 939GCCUCAGCUCAGGGUAGCC 428 939 CAGCAGUGUGAGGCCAGGG 250 939CAGCAGUGUGAGGCCAGGG 250 957 CCCUGGCCUCACACUGCUG 429 957GCAGAGUGAGACCCAGCCC 251 957 GCAGAGUGAGACCCAGCCC 251 975GGGCUGGGUCUCACUCUGC 430 975 CUCAUCCCGAGCACCUCCA 252 975CUCAUCCCGAGCACCUCCA 252 993 UGGAGGUGCUCGGGAUGAG 431 993ACAUCCUCCACGUUCUGCU 253 993 ACAUCCUCCACGUUCUGCU 253 1011AGCAGAACGUGGAGGAUGU 432 1011 UCAUCAUUCUCUGUCUCAU 254 1011UCAUCAUUCUCUGUCUCAU 254 1029 AUGAGACAGAGAAUGAUGA 433 1029UCCAUCAUCAUGUGUGUCC 255 1029 UCCAUCAUCAUGUGUGUCC 255 1047GGACACACAUGAUGAUGGA 434 1047 CACGACUGUCUCCAUGGCC 256 1047CACGACUGUCUCCAUGGCC 256 1065 GGCCAUGGAGACAGUCGUG 435 1065CCCGCAAAAGGACUCUCAG 257 1065 CCCGCAAAAGGACUCUCAG 257 1083CUGAGAGUCCUUUUGCGGG 436 1083 GGACCAAAGCUUUCAUGUA 258 1083GGACCAAAGCUUUCAUGUA 258 1101 UACAUGAAAGCUUUGGUCC 437 1101AAACUGUGCACCAAGCAGG 259 1101 AAACUGUGCACCAAGCAGG 259 1119CCUGCUUGGUGCACAGUUU 438 1119 GAAAUGAAAAUGUCUUGUG 260 1119GAAAUGAAAAUGUCUUGUG 260 1137 CACAAGACAUUUUCAUUUC 439 1137GUUACCUGAAAACACUGUG 261 1137 GUUACCUGAAAACACUGUG 261 1155CACAGUGUUUUCAGGUAAC 440 1155 GCACAUCUGUGUCUUGUUU 262 1155GCACAUCUGUGUCUUGUUU 262 1173 AAACAAGACACAGAUGUGC 441 1173UGGAAUAUUGUCCAUUGUC 263 1173 UGGAAUAUUGUCCAUUGUC 263 1191GACAAUGGACAAUAUUCCA 442 1191 CCAAUCCUAUGUUUUUGUU 264 1191CCAAUCCUAUGUUUUUGUU 264 1209 AACAAAAACAUAGGAUUGG 443 1209UCAAAGCCAGCGUCCUCCU 265 1209 UCAAAGCCAGCGUCCUCCU 265 1227AGGAGGACGCUGGCUUUGA 444 1227 UCUGUGACCAAUGUCUUGA 266 1227UCUGUGACCAAUGUCUUGA 266 1245 UCAAGACAUUGGUCACAGA 445 1245AUGCAUGCACUGUUCCCCC 267 1245 AUGCAUGCACUGUUCCCCC 267 1263GGGGGAACAGUGCAUGCAU 446 1263 CUGUGCAGCCGCUGAGCGA 268 1263CUGUGCAGCCGCUGAGCGA 268 1281 UCGCUCAGCGGCUGCACAG 447 1281AGGAGAUGCUCCUUGGGCC 269 1281 AGGAGAUGCUCCUUGGGCC 269 1299GGCCCAAGGAGCAUCUCCU 448 1299 CCUUUGAGUGCAGUCCUGA 270 1299CCUUUGAGUGCAGUCCUGA 270 1317 UCAGGACUGCACUCAAAGG 449 1317AUCAGAGCCGUGGUCCUUU 271 1317 AUCAGAGCCGUGGUCCUUU 271 1335AAAGGACCACGGCUCUGAU 450 1335 UGGGGUGAACUACCUUGGU 272 1335UGGGGUGAACUACCUUGGU 272 1353 ACCAAGGUAGUUCACCCCA 451 1353UUCCCCCACUGAUCACAAA 273 1353 UUCCCCCACUGAUCACAAA 273 1371UUUGUGAUCAGUGGGGGAA 452 1371 AAACAUGGUGGGUCCAUGG 274 1371AAACAUGGUGGGUCCAUGG 274 1389 CCAUGGACCCACCAUGUUU 453 1389GGCAGAGCCCAAGGGAAUU 275 1389 GGCAGAGCCCAAGGGAAUU 275 1407AAUUCCCUUGGGCUCUGCC 454 1407 UCGGUGUGCACCAGGGUUG 276 1407UCGGUGUGCACCAGGGUUG 276 1425 CAACCCUGGUGCACACCGA 455 1425GACCCCAGAGGAUUGCUGC 277 1425 GACCCCAGAGGAUUGCUGC 277 1443GCAGCAAUCCUCUGGGGUC 456 1443 CCCCAUCAGUGCUCCCUCA 278 1443CCCCAUCAGUGCUCCCUCA 278 1461 UGAGGGAGCACUGAUGGGG 457 1461ACAUGUCAGUACCUUCAAA 279 1461 ACAUGUCAGUACCUUCAAA 279 1479UUUGAAGGUACUGACAUGU 458 1479 ACUAGGGCCAAGCCCAGCA 280 1479ACUAGGGCCAAGCCCAGCA 280 1497 UGCUGGGCUUGGCCCUAGU 459 1497ACUGCUUGAGGAAAACAAG 281 1497 ACUGCUUGAGGAAAACAAG 281 1515CUUGUUUUCCUCAAGCAGU 460 1515 GCAUUCACAACUUGUUUUU 282 1515GCAUUCACAACUUGUUUUU 282 1533 AAAAACAAGUUGUGAAUGC 461 1533UGGUUUUUAAAACCCAGUC 283 1533 UGGUUUUUAAAACCCAGUC 283 1551GACUGGGUUUUAAAAACCA 462 1551 CCACAAAAUAACCAAUCCU 284 1551CCACAAAAUAACCAAUCCU 284 1569 AGGAUUGGUUAUUUUGUGG 463 1569UGGACAUGAAGAUUCUUUC 285 1569 UGGACAUGAAGAUUCUUUC 285 1587GAAAGAAUCUUCAUGUCCA 464 1587 CCCAAUUCACAUCUAACCU 286 1587CCCAAUUCACAUCUAACCU 286 1605 AGGUUAGAUGUGAAUUGGG 465 1605UCAUCUUCUUCACCAUUUG 287 1605 UCAUCUUCUUCACCAUUUG 287 1623CAAAUGGUGAAGAAGAUGA 466 1623 GGCAAUGCCAUCAUCUCCU 288 1623GGCAAUGCCAUCAUCUCCU 288 1641 AGGAGAUGAUGGCAUUGCC 467 1641UGCCUUCCUCCUGGGCCCU 289 1641 UGCCUUCCUCCUGGGCCCU 289 1659AGGGCCCAGGAGGAAGGCA 468 1659 UCUCUGCUCUGCGUGUCAC 290 1659UCUCUGCUCUGCGUGUCAC 290 1677 GUGACACGCAGAGCAGAGA 469 1677CCUGUGCUUCGGGCCCUUC 291 1677 CCUGUGCUUCGGGCCCUUC 291 1695GAAGGGCCCGAAGCACAGG 470 1695 CCCACAGGACAUUUCUCUA 292 1695CCCACAGGACAUUUCUCUA 292 1713 UAGAGAAAUGUCCUGUGGG 471 1713AAGAGAACAAUGUGCUAUG 293 1713 AAGAGAACAAUGUGCUAUG 293 1731CAUAGCACAUUGUUCUCUU 472 1731 GUGAAGAGUAAGUCAACCU 294 1731GUGAAGAGUAAGUCAACCU 294 1749 AGGUUGACUUACUCUUCAC 473 1749UGCCUGACAUUUGGAGUGU 295 1749 UGCCUGACAUUUGGAGUGU 295 1767ACACUCCAAAUGUCAGGCA 474 1767 UUCCCCUUCCACUGAGGGC 296 1767UUCCCCUUCCACUGAGGGC 296 1785 GCCCUCAGUGGAAGGGGAA 475 1785CAGUCGAUAGAGCUGUAUU 297 1785 CAGUCGAUAGAGCUGUAUU 297 1803AAUACAGCUCUAUCGACUG 476 1803 UAAGCCACUUAAAAUGUUC 298 1803UAAGCCACUUAAAAUGUUC 298 1821 GAACAUUUUAAGUGGCUUA 477 1821CACUUUUGACAAAGGCAAG 299 1821 CACUUUUGACAAAGGCAAG 299 1839CUUGCCUUUGUCAAAAGUG 478 1839 GCACUUGUGGGUUUUUGUU 300 1839GCACUUGUGGGUUUUUGUU 300 1857 AACAAAAACCCACAAGUGC 479 1857UUUGUUUUUCAUUCAGUCU 301 1857 UUUGUUUUUCAUUCAGUCU 301 1875AGACUGAAUGAAAAACAAA 480 1875 UUACGAAUACUUUUGCCCU 302 1875UUACGAAUACUUUUGCCCU 302 1893 AGGGCAAAAGUAUUCGUAA 481 1893UUUGAUUAAAGACUCCAGU 303 1893 UUUGAUUAAAGACUCCAGU 303 1911ACUGGAGUCUUUAAUCAAA 482 1911 UUAAAAAAAAUUUUAAUGA 304 1911UUAAAAAAAAUUUUAAUGA 304 1929 UCAUUAAAAUUUUUUUUAA 483 1929AAGAAAGUGGAAAACAAGG 305 1929 AAGAAAGUGGAAAACAAGG 305 1947CCUUGUUUUCCACUUUCUU 484 1947 GAAGUCAAAGCAAGGAAAC 306 1947GAAGUCAAAGCAAGGAAAC 306 1965 GUUUCCUUGCUUUGACUUC 485 1965CUAUGUAACAUGUAGGAAG 307 1965 CUAUGUAACAUGUAGGAAG 307 1983CUUCCUACAUGUUACAUAG 486 1983 GUAGGAAGUAAAUUAUAGU 308 1983GUAGGAAGUAAAUUAUAGU 308 2001 ACUAUAAUUUACUUCCUAC 487 2001UGAUGUAAUCUUGAAUUGU 309 2001 UGAUGUAAUCUUGAAUUGU 309 2019ACAAUUCAAGAUUACAUCA 488 2019 UAACUGUUCUUGAAUUUAA 310 2019UAACUGUUCUUGAAUUUAA 310 2037 UUAAAUUCAAGAACAGUUA 489 2037AUAAUCUGUAGGGUAAUUA 311 2037 AUAAUCUGUAGGGUAAUUA 311 2055UAAUUACCCUACAGAUUAU 490 2055 AGUAACAUGUGUUAAGUAU 312 2055AGUAACAUGUGUUAAGUAU 312 2073 AUACUUAACACAUGUUACU 491 2073UUUUCAUAAGUAUUUCAAA 313 2073 UUUUCAUAAGUAUUUCAAA 313 2091UUUGAAAUACUUAUGAAAA 492 2091 AUUGGAGCUUCAUGGCAGA 314 2091AUUGGAGCUUCAUGGCAGA 314 2109 UCUGCCAUGAAGCUCCAAU 493 2109AAGGCAAACCCAUCAACAA 315 2109 AAGGCAAACCCAUCAACAA 315 2127UUGUUGAUGGGUUUGCCUU 494 2127 AAAAUUGUCCCUUAAACAA 316 2127AAAAUUGUCCCUUAAACAA 316 2145 UUGUUUAAGGGACAAUUUU 495 2145AAAAUUAAAAUCCUCAAUC 317 2145 AAAAUUAAAAUCCUCAAUC 317 2163GAUUGAGGAUUUUAAUUUU 496 2163 CCAGCUAUGUUAUAUUGAA 318 2163CCAGCUAUGUUAUAUUGAA 318 2181 UUCAAUAUAACAUAGCUGG 497 2181AAAAAUAGAGCCUGAGGGA 319 2181 AAAAAUAGAGCCUGAGGGA 319 2199UCCCUCAGGCUCUAUUUUU 498 2199 AUCUUUACUAGUUAUAAAG 320 2199AUCUUUACUAGUUAUAAAG 320 2217 CUUUAUAACUAGUAAAGAU 499 2217GAUACAGAACUCUUUCAAA 321 2217 GAUACAGAACUCUUUCAAA 321 2235UUUGAAAGAGUUCUGUAUC 500 2235 AACCUUUUGAAAUUAACCU 322 2235AACCUUUUGAAAUUAACCU 322 2253 AGGUUAAUUUCAAAAGGUU 501 2253UCUCACUAUACCAGUAUAA 323 2253 UCUCACUAUACCAGUAUAA 323 2271UUAUACUGGUAUAGUGAGA 502 2271 AUUGAGUUUUCAGUGGGGC 324 2271AUUGAGUUUUCAGUGGGGC 324 2289 GCCCCACUGAAAACUCAAU 503 2289CAGUCAUUAUCCAGGUAAU 325 2289 CAGUCAUUAUCCAGGUAAU 325 2307AUUACCUGGAUAAUGACUG 504 2307 UCCAAGAUAUUUUAAAAUC 326 2307UCCAAGAUAUUUUAAAAUC 326 2325 GAUUUUAAAAUAUCUUGGA 505 2325CUGUCACGUAGAACUUGGA 327 2325 CUGUCACGUAGAACUUGGA 327 2343UCCAAGUUCUACGUGACAG 506 2343 AUGUACCUGCCCCCAAUCC 328 2343AUGUACCUGCCCCCAAUCC 328 2361 GGAUUGGGGGCAGGUACAU 507 2361CAUGAACCAAGACCAUUGA 329 2361 CAUGAACCAAGACCAUUGA 329 2379UCAAUGGUCUUGGUUCAUG 508 2379 AAUUCUUGGUUGAGGAAAC 330 2379AAUUCUUGGUUGAGGAAAC 330 2397 GUUUCCUCAACCAAGAAUU 509 2397CAAACAUGACCCUAAAUCU 331 2397 CAAACAUGACCCUAAAUCU 331 2415AGAUUUAGGGUCAUGUUUG 510 2415 UUGACUACAGUCAGGAAAG 332 2415UUGACUACAGUCAGGAAAG 332 2433 CUUUCCUGACUGUAGUCAA 511 2433GGAAUCAUUUCUAUUUCUC 333 2433 GGAAUCAUUUCUAUUUCUC 333 2451GAGAAAUAGAAAUGAUUCC 512 2451 CCUCCAUGGGAGAAAAUAG 334 2451CCUCCAUGGGAGAAAAUAG 334 2469 CUAUUUUCUCCCAUGGAGG 513 2469GAUAAGAGUAGAAACUGCA 335 2469 GAUAAGAGUAGAAACUGCA 335 2487UGCAGUUUCUACUCUUAUC 514 2487 AGGGAAAAUUAUUUGCAUA 336 2487AGGGAAAAUUAUUUGCAUA 336 2505 UAUGCAAAUAAUUUUCCCU 515 2505AACAAUUCCUCUACUAACA 337 2505 AACAAUUCCUCUACUAACA 337 2523UGUUAGUAGAGGAAUUGUU 516 2523 AAUCAGCUCCUUCCUGGAG 338 2523AAUCAGCUCCUUCCUGGAG 338 2541 CUCCAGGAAGGAGCUGAUU 517 2541GACUGCCCAGCUAAAGCAA 339 2541 GACUGCCCAGCUAAAGCAA 339 2559UUGCUUUAGCUGGGCAGUC 518 2559 AUAUGCAUUUAAAUACAGU 340 2559AUAUGCAUUUAAAUACAGU 340 2577 ACUGUAUUUAAAUGCAUAU 519 2577UCUUCCAUUUGCAAGGGAA 341 2577 UCUUCCAUUUGCAAGGGAA 341 2595UUCCCUUGCAAAUGGAAGA 520 2595 AAAGUCUCUUGUAAUCCGA 342 2595AAAGUCUCUUGUAAUCCGA 342 2613 UCGGAUUACAAGAGACUUU 521 2613AAUCUCUUUUUGCUUUCGA 343 2613 AAUCUCUUUUUGCUUUCGA 343 2631UCGAAAGCAAAAAGAGAUU 522 2631 AACUGCUAGUCAAGUGCGU 344 2631AACUGCUAGUCAAGUGCGU 344 2649 ACGCACUUGACUAGCAGUU 523 2649UCCACGAGCUGUUUACUAG 345 2649 UCCACGAGCUGUUUACUAG 345 2667CUAGUAAACAGCUCGUGGA 524 2667 GGGAUCCCUCAUCUGUCCC 346 2667GGGAUCCCUCAUCUGUCCC 346 2685 GGGACAGAUGAGGGAUCCC 525 2685CUCCGGGACCUGGUGCUGC 347 2685 CUCCGGGACCUGGUGCUGC 347 2703GCAGCACCAGGUCCCGGAG 526 2703 CCUCUACCUGACACUCCCU 348 2703CCUCUACCUGACACUCCCU 348 2721 AGGGAGUGUCAGGUAGAGG 527 2721UUGGGCUCCCUGUAACCUC 349 2721 UUGGGCUCCCUGUAACCUC 349 2739GAGGUUACAGGGAGCCCAA 528 2739 CUUCAGAGGCCCUCGCUGC 350 2739CUUCAGAGGCCCUCGCUGC 350 2757 GCAGCGAGGGCCUCUGAAG 529 2757CCAGCUCUGUAUCAGGACC 351 2757 CCAGCUCUGUAUCAGGACC 351 2775GGUCCUGAUACAGAGCUGG 530 2775 CCAGAGGAAGGGGCCAGAG 352 2775CCAGAGGAAGGGGCCAGAG 352 2793 CUCUGGCCCCUUCCUCUGG 531 2793GGCUCGUUGACUGGCUGUG 353 2793 GGCUCGUUGACUGGCUGUG 353 2811CACAGCCAGUCAACGAGCC 532 2811 GUGUUGGGAUUGAGUCUGU 354 2811GUGUUGGGAUUGAGUCUGU 354 2829 ACAGACUCAAUCCCAACAC 533 2829UGCCACGUGUUUGUGCUGU 355 2829 UGCCACGUGUUUGUGCUGU 355 2847ACAGCACAAACACGUGGCA 534 2847 UGGUGUGUCCCCCUCUGUC 356 2847UGGUGUGUCCCCCUCUGUC 356 2865 GACAGAGGGGGACACACCA 535 2865CCAGGCACUGAGAUACCAG 357 2865 CCAGGCACUGAGAUACCAG 357 2883CUGGUAUCUCAGUGCCUGG 536 2883 GCGAGGAGGCUCCAGAGGG 358 2883GCGAGGAGGCUCCAGAGGG 358 2901 CCCUCUGGAGCCUCCUCGC 537 2901GCACUCUGCUUGUUAUUAG 359 2901 GCACUCUGCUUGUUAUUAG 359 2919CUAAUAACAAGCAGAGUGC 538 2919 GAGAUUACCUCCUGAGAAA 360 2919GAGAUUACCUCCUGAGAAA 360 2937 UUUCUCAGGAGGUAAUCUC 539 2937AAAAGGUUCCGCUUGGAGC 361 2937 AAAAGGUUCCGCUUGGAGC 361 2955GCUCCAAGCGGAACCUUUU 540 2955 CAGAGGGGCUGAAUAGCAG 362 2955CAGAGGGGCUGAAUAGCAG 362 2973 CUGCUAUUCAGCCCCUCUG 541 2973GAAGGUUGCACCUCCCCCA 363 2973 GAAGGUUGCACCUCCCCCA 363 2991UGGGGGAGGUGCAACCUUC 542 2991 AACCUUAGAUGUUCUAAGU 364 2991AACCUUAGAUGUUCUAAGU 364 3009 ACUUAGAACAUCUAAGGUU 543 3009UCUUUCCAUUGGAUCUCAU 365 3009 UCUUUCCAUUGGAUCUCAU 365 3027AUGAGAUCCAAUGGAAAGA 544 3027 UUGGACCCUUCCAUGGUGU 366 3027UUGGACCCUUCCAUGGUGU 366 3045 ACACCAUGGAAGGGUCCAA 545 3045UGAUCGUCUGACUGGUGUU 367 3045 UGAUCGUCUGACUGGUGUU 367 3063AACACCAGUCAGACGAUCA 546 3063 UAUCACCGUGGGCUCCCUG 368 3063UAUCACCGUGGGCUCCCUG 368 3081 CAGGGAGCCCACGGUGAUA 547 3081GACUGGGAGUUGAUCGCCU 369 3081 GACUGGGAGUUGAUCGCCU 369 3099AGGCGAUCAACUCCCAGUC 548 3099 UUUCCCAGGUGCUACACCC 370 3099UUUCCCAGGUGCUACACCC 370 3117 GGGUGUAGCACCUGGGAAA 549 3117CUUUUCCAGCUGGAUGAGA 371 3117 CUUUUCCAGCUGGAUGAGA 371 3135UCUCAUCCAGCUGGAAAAG 550 3135 AAUUUGAGUGCUCUGAUCC 372 3135AAUUUGAGUGCUCUGAUCC 372 3153 GGAUCAGAGCACUCAAAUU 551 3153CCUCUACAGAGCUUCCCUG 373 3153 CCUCUACAGAGCUUCCCUG 373 3171CAGGGAAGCUCUGUAGAGG 552 3171 GACUCAUUCUGAAGGAGCC 374 3171GACUCAUUCUGAAGGAGCC 374 3189 GGCUCCUUCAGAAUGAGUC 553 3189CCCAUUCCUGGGAAAUAUU 375 3189 CCCAUUCCUGGGAAAUAUU 375 3207AAUAUUUCCCAGGAAUGGG 554 3207 UCCCUAGAAACUUCCAAAU 376 3207UCCCUAGAAACUUCCAAAU 376 3225 AUUUGGAAGUUUCUAGGGA 555 3225UCCCCUAAGCAGACCACUG 377 3225 UCCCCUAAGCAGACCACUG 377 3243CAGUGGUCUGCUUAGGGGA 556 3243 GAUAAAACCAUGUAGAAAA 378 3243GAUAAAACCAUGUAGAAAA 378 3261 UUUUCUACAUGGUUUUAUC 557 3261AUUUGUUAUUUUGCAACCU 379 3261 AUUUGUUAUUUUGCAACCU 379 3279AGGUUGCAAAAUAACAAAU 558 3279 UCGCUGGACUCUCAGUCUC 380 3279UCGCUGGACUCUCAGUCUC 380 3297 GAGACUGAGAGUCCAGCGA 559 3297CUGAGCAGUGAAUGAUUCA 381 3297 CUGAGCAGUGAAUGAUUCA 381 3315UGAAUCAUUCACUGCUCAG 560 3315 AGUGUUAAAUGUGAUGAAU 382 3315AGUGUUAAAUGUGAUGAAU 382 3333 AUUCAUCACAUUUAACACU 561 3333UACUGUAUUUUGUAUUGUU 383 3333 UACUGUAUUUUGUAUUGUU 383 3351AACAAUACAAAAUACAGUA 562 3351 UUCAAUUGCAUCUCCCAGA 384 3351UUCAAUUGCAUCUCCCAGA 384 3369 UCUGGGAGAUGCAAUUGAA 563 3369AUAAUGUGAAAAUGGUCCA 385 3369 AUAAUGUGAAAAUGGUCCA 385 3387UGGACCAUUUUCACAUUAU 564 3387 AGGAGAAGGCCAAUUCCUA 386 3387AGGAGAAGGCCAAUUCCUA 386 3405 UAGGAAUUGGCCUUCUCCU 565 3405AUACGCAGCGUGCUUUAAA 387 3405 AUACGCAGCGUGCUUUAAA 387 3423UUUAAAGCACGCUGCGUAU 566 3423 AAAAUAAAUAAGAAACAAC 388 3423AAAAUAAAUAAGAAACAAC 388 3441 GUUGUUUCUUAUUUAUUUU 567 3441CUCUUUGAGAAACAACAAU 389 3441 CUCUUUGAGAAACAACAAU 389 3459AUUGUUGUUUCUCAAAGAG 568 3459 UUUCUACUUUGAAGUCAUA 390 3459UUUCUACUUUGAAGUCAUA 390 3477 UAUGACUUCAAAGUAGAAA 569 3477ACCAAUGAAAAAAUGUAUA 391 3477 ACCAAUGAAAAAAUGUAUA 391 3495UAUACAUUUUUUCAUUGGU 570 3495 AUGCACUUAUAAUUUUCCU 392 3495AUGCACUUAUAAUUUUCCU 392 3513 AGGAAAAUUAUAAGUGCAU 571 3513UAAUAAAGUUCUGUACUCA 393 3513 UAAUAAAGUUCUGUACUCA 393 3531UGAGUACAGAACUUUAUUA 572 3531 AAAUGUAGCCACCAAAAAA 394 3531AAAUGUAGCCACCAAAAAA 394 3549 UUUUUUGGUGGCUACAUUU 573 3540CACCAAAAAAAAAAAAAAA 395 3540 CACCAAAAAAAAAAAAAAA 395 3558UUUUUUUUUUUUUUUGGUG 574 The 3′-ends of the Upper sequence and the Lowersequence of the siNA construct can include an overhang sequence, forexample about 1, 2, 3, or 4 nucleotides in length, preferably 2nucleotides in length, wherein the overhanging sequence of the lowersequence is optionally complementary to a portion of the targetsequence. The upper sequence is also referred to as the sense strand,whereas the lower sequence is also referred to as the antisense strand.The upper and lower sequences in the Table can further comprise achemical modification having Formulae I-VII, such as exemplary siNAconstructs shown in FIGS. 4 and 5, or having modifications described inTable IV or any combination thereof.

TABLE III SDF-1 Synthetic Modified siNA constructs Target Seq Seq PosTarget ID Cmpd# Aliases Sequence ID 190 GAUUCUUCGAAAGCCAUGUUGCC 575CXCL12b:192U21 sense siNA UUCUUCGAAAGCCAUGUUGTT 599 213AGAGCCAACGUCAAGCAUCUCAA 576 CXCL12b:215U21 sense siNAAGCCAACGUCAAGCAUCUCTT 600 214 GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:216U21sense siNA GCCAACGUCAAGCAUCUCATT 601 215 AGCCAACGUCAAGCAUCUCAAAA 578CXCL12b:217U21 sense siNA CCAACGUCAAGCAUCUCAATT 602 216GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:218U21 sense siNACAACGUCAAGCAUCUCAAATT 603 217 CCAACGUCAAGCAUCUCAAAAUU 580 CXCL12b:219U21sense siNA AACGUCAAGCAUCUCAAAATT 604 218 CAACGUCAAGCAUCUCAAAAUUC 581CXCL12b:220U21 sense siNA ACGUCAAGCAUCUCAAAAUTT 605 219AACGUCAAGCAUCUCAAAAUUCU 582 CXCL12b:221U21 sense siNACGUCAAGCAUCUCAAAAUUTT 606 998 CUCCACGUUCUGCUCAUCAUUCU 583CXCL12b:1000U21 sense siNA CCACGUUCUGCUCAUCAUUTT 607 1000CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1002U21 sense siNAACGUUCUGCUCAUCAUUCUTT 608 1496 CACUGCUUGAGGAAAACAAGCAU 585CXCL12b:1498U21 sense siNA CUGCUUGAGGAAAACAAGCTT 609 1821CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1823U21 sense siNACUUUUGACAAAGGCAAGCATT 610 2109 AAGGCAAACCCAUCAACAAAAAU 587CXCL12b:2111U21 sense siNA GGCAAACCCAUCAACAAAATT 611 2110AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2112U21 sense siNAGCAAACCCAUCAACAAAAATT 612 2116 ACCCAUCAACAAAAAUUGUCCCU 589CXCL12b:2118U21 sense siNA CCAUCAACAAAAAUUGUCCTT 613 2631AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2633U21 sense siNACUGCUAGUCAAGUGCGUCCTT 614 456 CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:458U21sense siNA UAACCAUGAGGACCAGGUGTT 615 590 UACCUGUGCACGUUGGAACUUUU 592CXCL12a:592U21 sense siNA CCUGUGCACGUUGGAACUUTT 616 807GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:809U21 sense siNAUUAACAGGGAGCUGGAAAATT 617 968 GGGCUCCAUGUAGAAGCCACUAU 594 CXCL22a:970U21sense siNA GCUCCAUGUAGAAGCCACUTT 618 991 UACUGGGACUGUGCUCAGAGACC 595CXCL12a:993U21 sense siNA CUGGGACUGUGCUCAGAGATT 619 1021CAGCUAUUCCUACUCUCUCCCCG 596 CXCL12a:1023U21 sense siNAGCUAUUCCUACUCUCUCCCTT 620 1321 GAGUGACUGGGUUUUGUGAUUGC 597CXCL12a:1323U21 sense siNA GUGACUGGGUUUUGUGAUUTT 621 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1342U21 sense siNAGCCUCUGAAGCCUAUGUAUTT 622 190 GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:210L21antisense siNA CAACAUGGCUUUCGAAGAATT 623 (192C) 213AGAGCCAACGUCAAGCAUCUCAA 576 CXCL12b:233L21 antisense siNAGAGAUGCUUGACGUUGGCUTT 624 (215C) 214 GAGCCAACGUCAAGCAUCUCAAA 577CXCL12b:234L21 antisense siNA UGAGAUGCUUGACGUUGGCTT 625 (216C) 215AGCCAACGUCAAGCAUCUCAAAA 578 CXCL12b:235L21 antisense siNAUUGAGAUGCUUGACGUUGGTT 626 (217C) 216 GCCAACGUCAAGCAUCUCAAAAU 579CXCL12b:236L21 antisense siNA UUUGAGAUGCUUGACGUUGTT 627 (218C) 217CCAACGUCAAGCAUCUCAAAAUU 580 CXCL12b:237L21 antisense siNAUUUUGAGAUGCUUGACGUUTT 628 (219C) 218 CAACGUCAAGCAUCUCAAAAUUC 581CXCL12b:238L21 antisense siNA AUUUUGAGAUGCUUGACGUTT 629 (220C) 219AACGUCAAGCAUCUCAAAAUUCU 582 CXCL12b:239L21 antisense siNAAAUUUUGAGAUGCUUGACGTT 630 (221C) 998 CUCCACGUUCUGCUCAUCAUUCU 583CXCL12b:1018L21 antisense AAUGAUGAGCAGAACGUGGTT 631 siNA (1000C) 1000CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1020L21 antisenseAGAAUGAUGAGCAGAACGUTT 632 siNA (1002C) 1496 CACUGCUUGAGGAAAACAAGCAU 585CXCL12b:1516L21 antisense GCUUGUUUUCCUCAAGCAGTT 633 siNA (1498C) 1821CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1841L21 antisenseUGCUUGCCUUUGUCAAAAGTT 634 siNA (1823C) 2109 AAGGCAAACCCAUCAACAAAAAU 587CXCL12b:2129L21 antisense UUUUGUUGAUGGGUUUGCCTT 635 siNA (2111C) 2110AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2130L21 antisenseUUUUUGUUGAUGGGUUUGCTT 636 siNA (2112C) 2116 ACCCAUCAACAAAAAUUGUCCCU 589CXCL12b:2136L21 antisense GGACAAUUUUUGUUGAUGGTT 637 siNA (2118C) 2631AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2651L21 antisenseGGACGCACUUGACUAGCAGTT 638 siNA (2633C) 456 CUUAACCAUGAGGACCAGGUGUG 591CXCL12a:476L21 antisense siNA CACCUGGUCCUCAUGGUUATT 639 (458C) 590UACCUGUGCACGUUGGAACUUUU 592 CXCL12a:610L21 antisense siNAAAGUUCCAACGUGCACAGGTT 640 (592C) 807 GCUUAACAGGGAGCUGGAAAAAG 593CXCL12a:827L21 antisense siNA UUUUCCAGCUCCCUGUUAATT 641 (809C) 968GGGCUCCAUGUAGAAGCCACUAU 594 CXCL12a:988L21 antisense siNAAGUGGCUUCUACAUGGAGCTT 642 (970C) 991 UACUGGGACUGUGCUCAGAGACC 595CXCL12a:1011L21 antisense UCUCUGAGCACAGUCCCAGTT 643 siNA (993C) 1021CAGCUAUUCCUACUCUCUCCCCG 596 CXCL12a:1041L21 antisenseGGGAGAGAGUAGGAAUAGCTT 644 siNA (1023C) 1321 GAGUGACUGGGUUUUGUGAUUGC 597CXCL12a:1341L21 antisense AAUCACAAAACCCAGUCACTT 645 siNA (1323C) 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1360L21 antisenseAUACAUAGGCUUCAGAGGCTT 646 siNA (1342C) 190 GAUUCUUCGAAAGCCAUGUUGCC 575CXCL12b:192U21 sense siNA B uucuucGAAAGccAuGuuGTT B 647 stab04 213AGAGCCAACGUCAAGCAUCUCAA 576 CXCL12b:215U21 sense siNA BAGccAAcGucAAGcAucucTT B 648 stab04 214 GAGCCAACGUCAAGCAUCUCAAA 577CXCL12b:216U21 sense siNA B GccAAcGucAAGcAucucATT B 649 stab04B 215AGCCAACGUCAAGCAUCUCAAAA 578 CXCL12b:217U21 sense siNA BccAAcGucAAGcAucucAATT B 650 stab04B 216 GCCAACGUCAAGCAUCUCAAAAU 579CXCL12b:218U21 sense siNA B cAAcGucAAGcAucucAAATT B 651 stab04B 217CCAACGUCAAGCAUCUCAAAAUU 580 CXCL12b:219U21 sense siNA BAAcGucAAGcAucucAAAATT B 652 stab04 218 CAACGUCAAGCAUCUCAAAAUUC 581CXCL12b:220U21 sense siNA B AcGucAAGcAucucAAAAuTT B 653 stab04 219AACGUCAAGCAUCUCAAAAUUCU 582 CXCL12b:221U21 sense siNA BcGucAAGcAucucAAAAuuTT B 654 stab04 998 CUCCACGUUCUGCUCAUCAUUCU 583CXCL12b:1000U21 sense siNA B ccAcGuucuGcucAucAuuTT B 655 stab04 1000CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1002U21 sense siNA BAcGuucuGcucAucAuucuTT B 656 stab04 1496 CACUGCUUGAGGAAAACAAGCAU 585CXCL12b:1498U21 sense siNA B cuGcuuGAGGAAAAcAAGcTT B 657 stab04 1821CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1823U21 sense siNA BcuuuuGAcAAAGGcAAGcATT B 658 stab04 2109 AAGGCAAACCCAUCAACAAAAAU 587CXCL12b:2111U21 sense siNA B GGcAAAcccAucAAcAAAATT B 659 stab04 2110AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2112U21 sense siNA BGcAAAcccAucAAcAAAAATT B 660 stab04 2116 ACCCAUCAACAAAAAUUGUCCCU 589CXCL12b:2118U21 sense siNA B ccAucAAcAAAAAuuGuccTT B 661 stab04 2631AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2633U21 sense siNA BcuGcuAGucAAGuGcGuccTT B 662 stab04 456 CUUAACCAUGAGGACCAGGUGUG 591CXCL12a:458U21 sense siNA B uAAccAuGAGGAccAGGuGTT B 663 stab04 590UACCUGUGCACGUUGGAACUUUU 592 CXCL12a:592U21 sense siNA BccuGuGcAcGuuGGAAcuuTT B 664 stab04 807 GCUUAACAGGGAGCUGGAAAAAG 593CXCL12a:809U21 sense siNA B uuAAcAGGGAGcuGGAAAATT B 665 stab04 968GGGCUCCAUGUAGAAGCCACUAU 594 CXCL12a:970U21 sense siNA BGcuccAuGuAGAAGccAcuTT B 666 stab04 991 UACUGGGACUGUGCUCAGAGACC 595CXCL12a:993U21 sense siNA B cuGGGAcuGuGcucAGAGATT B 667 stab04 1021CAGCUAUUCCUACUCUCUCCCCG 596 CXCL12a:1023U21 sense siNA BGcuAuuccuAcucucucccTT B 668 stab04 1321 GAGUGACUGGGUUUUGUGAUUGC 597CXCL12a:1323U21 sense siNA B GuGAcuGGGuuuuGuGAuuTT B 669 stab04 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1342U21 sense siNA BGccucuGAAGccuAuGuAuTT B 670 stab04 190 GAUUCUUCGAAAGCCAUGUUGCC 575CXCL12b:210L21 antisense siNA cAAcAuGGcuuucGAAGAATsT 671 (192C) stab05213 AGAGCCAACGUCAAGCAUCUCAA 576 CXCL12b:233L21 antisense siNAGAGAuGcuuGAcGuuGGcuTsT 672 (215C) stab05 214 GAGCCAACGUCAAGCAUCUCAAA 577CXCL12b:234L21 antisense siNA uGAGAuGcuuGAcGuuGGcTsT 673 (216C) stab05215 AGCCAACGUCAAGCAUCUCAAAA 578 CXCL12b:235L21 antisense siNAuuGAGAuGcuuGAcGuuGGTsT 674 (217C) stab05 216 GCCAACGUCAAGCAUCUCAAAAU 579CXCL12b:236L21 antisense siNA uuuGAGAuGcuuGAcGuuGTsT 675 (218C) stab05217 CCAACGUCAAGCAUCUCAAAAUU 580 CXCL12b:237L21 antisense siNAuuuuGAGAuGcuuGAcGuuTsT 676 (219C) stab05 218 CAACGUCAAGCAUCUCAAAAUUC 581CXCL12b:238L21 antisense siNA AuuuuGAGAuGcuuGAcGuTsT 677 (220C) stab05219 AACGUCAAGCAUCUCAAAAUUCU 582 CXCL12b:239L21 antisense siNAAAuuuuGAGAuGcuuGAcGTsT 678 (221C) stab05 998 CUCCACGUUCUGCUCAUCAUUCU 583CXCL12b:1018L21 antisense siNA AAuGAuGAGcAGAAcGuGGTsT 679 (1000C) stab051000 CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1020L21 antisense siNAAGAAuGAuGAGcAGAAcGuTsT 680 (1002C) stab05 1496 CACUGCUUGAGGAAAACAAGCAU585 CXCL12b:1516L21 antisense siNA GcuuGuuuuccucAAGcAGTsT 681 (1498C)stab05 1821 CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1841L21 antisense siNAuGcuuGccuuuGucAAAAGTsT 682 (1823C) stab05 2109 AAGGCAAACCCAUCAACAAAAAU587 CXCL12b:2129L21 antisense siNA uuuuGuuGAuGGGuuuGccTsT 683 (2111C)stab05 2110 AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2130L21 antisense siNAuuuuuGuuGAuGGGuuuGcTsT 684 (2112C) stab05 2116 ACCCAUCAACAAAAAUUGUCCCU589 CXCL12b:2136L21 antisense siNA GGAcAAuuuuuGuuGAuGGTsT 685 (2118C)stab05 2631 AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2651L21 antisense siNAGGAcGcAcuuGAcuAGcAGTsT 686 (2633C) stab05 456 CUUAACCAUGAGGACCAGGUGUG591 CXCL12a:476L21 antisense siNA cAccuGGuccucAuGGuuATsT 687 (458C)stab05 590 UACCUGUGCACGUUGGAACUUUU 592 CXCL12a:610L21 antisense siNAAAGuuccAAcGuGcAcAGGTsT 688 (592C) stab05 807 GCUUAACAGGGAGCUGGAAAAAG 593CXCL12a:827L21 antisense siNA uuuuccAGcucccuGuuAATsT 689 (809C) stab05968 GGGCUCCAUGUAGAAGCCACUAU 594 CXCL12a:988L21 antisense siNAAGuGGcuucuAcAuGGAGcTsT 690 (970C) stab05 991 UACUGGGACUGUGCUCAGAGACC 595CXCL12a:1011L21 antisense ucucuGAGcAcAGucccAGTsT 691 siNA (993C) stab051021 CAGCUAUUCCUACUCUCUCCCCG 596 CXCLl2a:1041L21 antisenseGGGAGAGAGuAGGAAuAGcTsT 692 siNA (1023C) stab05 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1341L21 antisenseAAucAcAAAAcccAGucAcTsT 693 siNA (1323C) stab05 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1360L21 antisenseAuAcAuAGGcuucAGAGGcTsT 694 siNA (1342C) stab05 190GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:192U21 sense siNA BuucuucGAAAGccAuGuuGTT B 695 stab07 213 AGAGCCAACGUCAAGCAUCUCAA 576CXCL12b:215U21 sense siNA B AGccAAcGucAAGcAucucTT B 696 stab07 214GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:216U21 sense siNA BGccAAcGucAAGcAucucATT B 697 stab07 215 AGCCAACGUCAAGCAUCUCAAAA 578CXCL12b:217U21 sense siNA B ccAAcGucAAGcAucucAATT B 698 stab07 216GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:218U21 sense siNA BcAAcGucAAGcAucucAAATT B 699 stab07 217 CCAACGUCAAGCAUCUCAAAAUU 580CXCL12b:219U21 sense siNA B AAcGucAAGcAucucAAAATT B 700 stab07 218CAACGUCAAGCAUCUCAAAAUUC 581 CXCL12b:220U21 sense siNA BAcGucAAGcAucucAAAAuTT B 701 stab07 219 AACGUCAAGCAUCUCAAAAUUCU 582CXCL12b:221U21 sense siNA B cGucAAGcAucucAAAAuuTT B 702 stab07 998CUCCACGUUCUGCUCAUCAUUCU 583 CXCL12b:1000U21 sense siNA BccAcGuucuGcucAucAuuTT B 703 stab07 1000 CCACGUUCUGCUCAUCAUUCUCU 584CXCL12b:1002U21 sense siNA B AcGuucuGcucAucAuucuTT B 704 stab07 1496CACUGCUUGAGGAAAACAAGCAU 585 CXCL12b:1498U21 sense siNA BcuGcuuGAGGAAAAcAAGcTT B 705 stab07 1821 CACUUUUGACAAAGGCAAGCACU 586CXCL12b:1823U21 sense siNA B cuuuuGAcAAAGGcAAGcATT B 706 stab07 2109AAGGCAAACCCAUCAACAAAAAU 587 CXCL12b:2111U21 sense siNA BGGcAAAcccAucAAcAAAATT B 707 stab07 2110 AGGCAAACCCAUCAACAAAAAUU 588CXCL12b:2112U21 sense siNA B GcAAAcccAucAAcAAAAATT B 708 stab07 2116ACCCAUCAACAAAAAUUGUCCCU 589 CXCL12b:2118U21 sense siNA BccAucAAcAAAAAuuGuccTT B 709 stab07 2631 AACUGCUAGUCAAGUGCGUCCAC 590CXCL12b:2633U21 sense siNA B cuGcuAGucAAGuGcGuccTT B 710 stab07 456CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:458U21 sense siNA BuAAccAuGAGGAccAGGuGTT B 711 stab07 590 UACCUGUGCACGUUGGAACUUUU 592CXCL12a:592U21 sense siNA B ccuGuGcAcGuuGGAAcuuTT B 712 stab07 807GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:809U21 sense siNA BuuAAcAGGGAGcuGGAAAATT B 713 stab07 968 GGGCUCCAUGUAGAAGCCACUAU 594CXCL12a:970U21 sense siNA B GcuccAuGuAGAAGccAcuTT B 714 stab07 991UACUGGGACUGUGCUCAGAGACC 595 CXCL12a:993U21 sense siNA BcuGGGAcuGuGcucAGAGATT B 715 stab07 1021 CAGCUAUUCCUACUCUCUCCCCG 596CXCL12a:1023U21 sense siNA B GcuAuuccuAcucucucccTT B 716 stab07 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1323U21 sense siNA BGuGAcuGGGuuuuGuGAuuTT B 717 stab07 1340 UUGCCUCUGAAGCCUAUGUAUGC 598CXCL12a:1342U21 sense siNA B GccucuGAAGccuAuGuAuTT B 718 stab07 190GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:210L21 antisense siNAcAAcAuGGcuuucGAAGAATsT 719 (192C) stab11 213 AGAGCCAACGUCAAGCAUCUCAA 576CXCL12b:233L21 antisense siNA GAGAuGcuuGAcGuuGGcuTsT 720 (215C) stab11214 GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:234L21 antisense siNAuGAGAuGcuuGAcGuuGGcTsT 721 (216C) stab11 215 AGCCAACGUCAAGCAUCUCAAAA 578CXCL12b:235L21 antisense siNA uuGAGAuGcuuGAcGuuGGTsT 722 (217C) stab11216 GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:236L21 antisense siNAuuuGAGAuGcuuGAcGuuGTsT 723 (218C) stab11 217 CCAACGUCAAGCAUCUCAAAAUU 580CXCL12b:237L21 antisense siNA uuuuGAGAuGcuuGAcGuuTsT 724 (219C) stab11218 CAACGUCAAGCAUCUCAAAAUUC 581 CXCL12b:238L21 antisense siNAAuuuuGAGAuGcuuGAcGuTsT 725 (220C) stab11 219 AACGUCAAGCAUCUCAAAAUUCU 582CXCL12b:239L21 antisense siNA AAuuuuGAGAuGcuuGAcGTsT 726 (221C) stab11998 CUCCACGUUCUGCUCAUCAUUCU 583 CXCL12b:1018L21 antisenseAAuGAuGAGcAGAAcGuGGTsT 727 siNA (1000C) stab11 1000CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1020L21 antisenseAGAAuGAuGAGcAGAAcGuTsT 728 siNA (1002C) stab11 1496CACUGCUUGAGGAAAACAAGCAU 585 CXCL12b:1516L21 antisenseGcuuGuuuuccucAAGcAGTsT 729 siNA (1498C) stab11 1821CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1841L21 antisenseUGcuuGccuuuGucAAAAGTsT 730 siNA (1823C) stab11 2109AAGGCAAACCCAUCAACAAAAAU 587 CXCL12b:2129L21 antisenseuuuuGuuGAuGGGuuuGccTsT 731 siNA (2111C) stab11 2110AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2130L21 antisenseuuuuuGuuGAuGGGuuuGcTsT 732 siNA (2112C) stab11 2116ACCCAUCAACAAAAAUUGUCCCU 589 CXCL12b:2136L21 antisenseGGAcAAuuuuuGuuGAuGGTsT 733 siNA (2118C) stab11 2631AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2651L21 antisenseGGAcGcAcuuGAcuAGcAGTsT 734 siNA (2633C) stab11 456CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:476L21 antisense siNAcAccuGGuccucAuGGuuATsT 735 (458C) stab11 590 UACCUGUGCACGUUGGAACUUUU 592CXCL12a:610L21 antisense siNA AAGuuccAAcGuGcAcAGGTsT 736 (592C) stab11807 GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:827L21 antisense siNAuuuuccAGcucccuGuuAATsT 737 (809C) stab11 968 GGGCUCCAUGUAGAAGCCACUAU 594CXCL12a:988L21 antisense siNA AGuGGcuucuAcAuGGAGcTsT 738 (970C) stab11991 UACUGGGACUGUGCUCAGAGACC 595 CXCL12a:1011L21 antisenseucucuGAGcAcAGucccAGTsT 739 siNA (993C) stab11 1021CAGCUAUUCCUACUCUCUCCCCG 596 CXCL12a:1041L21 antisenseGGGAGAGAGuAGGAAuAGcTsT 740 siNA (1023C) stab11 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1341L21 antisenseAAucAcAAAAcccAGucAcTsT 741 siNA (1323C) stab11 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1360L21 antisenseAuAcAuAGGcuucAGAGGcTsT 742 (siNA 1342C) stab11 190GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:192U21 sense siNA BuucuucGAAAGccAuGuuGTT B 743 stab18 213 AGAGCCAACGUCAAGCAUCUCAA 576CXCL12b:215U21 sense siNA B AGccAAcGucAAGcAucucTT B 744 stab18 214GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:216U21 sense siNA BGccAAcGucAAGcAucucATT B 745 stab18 215 AGCCAACGUCAAGCAUCUCAAAA 578CXCL12b:217U21 sense siNA B ccAAcGucAAGcAucucAATT B 746 stab18 216GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:218U21 sense siNA BcAAcGucAAGcAucucAAATT B 747 stab18 217 CCAACGUCAAGCAUCUCAAAAUU 580CXCL12b:219U21 sense siNA B AAcGucAAGcAucucAAAATT B 748 stab18 218CAACGUCAAGCAUCUCAAAAUUC 581 CXCL12b:220U21 sense siNA BAcGucAAGcAucucAAAAuTT B 749 stab18 219 AACGUCAAGCAUCUCAAAAUUCU 582CXCL12b:221U21 sense siNA B cGucAAGcAucucAAAAuuTT B 750 stab18 998CUCCACGUUCUGCUCAUCAUUCU 583 CXCL12b:1000U21 sense siNA BccAcGuucuGcucAucAuuTT B 751 stab18 1000 CCACGUUCUGCUCAUCAUUCUCU 584CXCL12b:1002U21 sense siNA B AcGuucuGcucAucAuucuTT B 752 stab18 1496CACUGCUUGAGGAAAACAAGCAU 585 CXCL12b:1498U21 sense siNA BcuGcuuGAGGAAAAcAAGcTT B 753 stab18 1821 CAOUUUUGACAAAGGCAAGCACU 586CXCL12b:1823U21 sense siNA B cuuuuGAcAAAGGcAAGcATT B 754 stab18 2109AAGGCAAACCCAUCAACAAAAAU 587 CXCL12b:2111U21 sense siNA BGGcAAAcccAucAAcAAAATT B 755 stab18 2110 AGGCAAACCCAUCAACAAAAAUU 588CXCL12b:2112U21 sense siNA B GcAAAcccAucAAcAAAAATT B 756 stab18 2116ACCCAUCAACAAAAAUUGUCCCU 589 CXCL12b:2118U21 sense siNA BccAucAAcAAAAAuuGuccTT B 757 stab18 2631 AACUGCUAGUCAAGUGCGUCCAC 590CXCL12b:2633U21 sense siNA B cuGcuAGucAAGuGcGuccTT B 758 stab18 456CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:458U21 sense siNA BuAAccAuGAGGAccAGGuGTT B 759 stab18 590 UACCUGUGCACGUUGGAACUUUU 592CXCL12a:592U21 sense siNA B ccuGuGcAcGuuGGAAcuuTT B 760 stab18 807GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:809U21 sense siNA BuuAAcAGGGAGcuGGAAAATT B 761 stab18 968 GGGCUCCAUGUAGAAGCCACUAU 594CXCL12a:970U21 sense siNA B GcuccAuGuAGAAGccAcuTT B 762 stab18 991UACUGGGACUGUGCUCAGAGACC 595 CXCL12a:993U21 sense siNA BcuGGGAcuGuGcucAGAGATT B 763 stab18 1021 CAGCUAUUCCUACUCUCUCCCCG 596CXCL12a:1023U21 sense siNA B GcuAuuccuAcucucucccTT B 764 stab18 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1323U21 sense siNA BGuGAcuGGGuuuuGuGAuuTT B 765 stab18 1340 UUGCCUCUGAAGCCUAUGUAUGC 598CXCL12a:1342U21 sense siNA B GccucuGAAGccuAuGuAuTT B 766 stab18 190GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:210L21 antisense siNAcAAcAuGGcuuucGAAGAATsT 767 (192C) stab08 213 AGAGCCAACGUCAAGCAUCUCAA 576CXCL12b:233L21 antisense siNA GAGAuGcuuGAcGuuGGcuTsT 768 (215C) stab08214 GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:234L21 antisense siNAuGAGAuGcuuGAcGuuGGcTsT 769 (216C) stab08 215 AGCCAACGUCAAGCAUCUCAAAA 578CXCL12b:235L21 antisense siNA uuGAGAuGcuuGAcGuuGGTsT 770 (217C) stab08216 GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:236L21 antisense siNAuuuGAGAuGcuuGAcGuuGTsT 771 (218C) stab08 217 CCAACGUCAAGCAUCUCAAAAUU 580CXCL12b:237L21 antisense siNA uuuuGAGAuGcuuGAcGuuTsT 772 (219C) stab08218 CAACGUCAAGCAUCUCAAAAUUC 581 CXCL12b:238L21 antisense siNAAuuuuGAGAuGcuuGAcGuTsT 773 (220C) stab08 219 AACGUCAAGCAUCUCAAAAUUCU 582CXCL12b:239L21 antisense siNA AAuuuuGAGAuGcuuGAcGTsT 774 (221C) stab08998 CUCCACGUUCUGCUCAUCAUUCU 583 CXCL12b:1018L21 antisenseAAuGAuGAGcAGAAcGuGGTsT 775 siNA (1000C) stab08 1000CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1020L21 antisenseAGAAuGAuGAGcAGAAcGuTsT 776 siNA (1002C) stab08 1496CACUGCUUGAGGAAAACAAGCAU 585 CXCL12b:1516L21 antisenseGcuuGuuuuccucAAGcAGTsT 777 siNA (1498C) stab08 1821CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1841L21 antisenseuGcuuGccuuuGucAAAAGTsT 778 siNA (1823C) stab08 2109AAGGCAAACCCAUCAACAAAAAU 587 CXCL12b:2129L21 antisenseuuuuGuuGAuGGGuuuGccTsT 779 siNA (2111C) stab08 2110AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2130L21 antisenseuuuuuGuuGAuGGGuuuGcTsT 780 siNA (2112C) stab08 2116ACCCAUCAACAAAAAUUGUCCCU 589 CXCL12b:2136L21 antisenseGGAcAAuuuuuGuuGAuGGTsT 781 siNA (2118C) stab08 2631AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2651L21 antisenseGGAcGcAcuuGAcuAGcAGTsT 782 siNA (2633C) stab08 456CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:476L21 antisense siNAcAccuGGuccucAuGGuuATsT 783 (458C) stab08 590 UACCUGUGCACGUUGGAACUUUU 592CXCL12a:610L21 antisense siNA AAGuuccAAcGuGcAcAGGTsT 784 (592C) stab08807 GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:827L21 antisense siNAuuuuccAGcucccuGuuAATsT 785 (809C) stab08 968 GGGCUCCAUGUAGAAGCCACUAU 594CXCL12a:988L21 antisense siNA AGuGGcuucuAcAuGGAGcTsT 786 (970C) stab08991 UACUGGGACUGUGCUCAGAGACC 595 CXCL12a:1011L21 antisenseucucuGAGcAcAGucccAGTsT 787 siNA (993C) stab08 1021CAGCUAUUCCUACUCUCUCCCCG 596 CXCLl2a:1041L21 antisenseGGGAGAGAGuAGGAAuAGcTsT 788 siNA (1023C) stab08 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1341L21 antisenseAAucAcAAAAcccAGucAcTsT 789 siNA (1323C) stab08 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1360L21 antisenseAuAcAuAGGcuucAGAGGcTsT 790 siNA (1342C) stab08 190GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:192U21 sense siNA BUUCUUCGAAAGCCAUGUUGTT B 791 stab09 213 AGAGCCAACGUCAAGCAUCUCAA 576CXCL12b:215U21 sense siNA B AGCCAACGUCAAGCAUCUCTT B 792 stab09 214GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:216U21 sense siNA BGCCAACGUCAAGCAUCUCATT B 793 stab09 215 AGCCAACGUCAAGCAUCUCAAAA 578CXCL12b:217U21 sense siNA B CCAACGUCAAGCAUCUCAATT B 794 stab09 216GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:218U21 sense siNA BCAACGUCAAGCAUCUCAAATT B 795 stab09 217 CCAACGUCAAGCAUCUCAAAAUU 580CXCL12b:219U21 sense siNA B AACGUCAAGCAUCUCAAAATT B 796 stab09 218CAACGUCAAGCAUCUCAAAAUUC 581 CXCL12b:220U21 sense siNA BACGUCAAGCAUCUCAAAAUTT B 797 stab09 219 AACGUCAAGCAUCUCAAAAUUCU 582CXCL12b:221U21 sense siNA B GGUGAAGGAUGUGAAAAUUTT B 798 stab09 998CUCCACGUUCUGCUCAUCAUUCU 583 CXCL12b:1000U21 sense siNA BCCACGUUCUGCUCAUCAUUTT B 799 stab09 1000 CCACGUUCUGCUCAUCAUUCUCU 584CXCL12b:1002U21 sense siNA B ACGUUCUGCUCAUCAUUCUTT B 800 stab09 1496CACUGCUUGAGGAAAACAAGCAU 585 CXCL12b:1498U21 sense siNA BCUGCUUGAGGAAAACAAGCTT B 801 stab09 1821 CACUUUUGACAAAGGCAAGCACU 586CXCL12b:1823U21 sense siNA B CUUUUGACAAAGGCAAGCATT B 802 stab09 2109AAGGCAAACCCAUCAACAAAAAU 587 CXCL12b:2111U21 sense siNA BGGCAAACCCAUCAACAAAATT B 803 stab09 2110 AGGCAAACCCAUCAACAAAAAUU 588CXCL12b:2112U21 sense siNA B GCAAACCCAUCAACAAAAATT B 804 stab09 2116ACCCAUCAACAAAAAUUGUCCCU 589 CXCL12b:2118U21 sense siNA BCCAUCAACAAAAAUUGUCCTT B 805 stab09 2631 AACUGCUAGUCAAGUGCGUCCAC 590CXCL12b:2633U21 sense siNA B CUGCUAGUCAAGUGCGUCCTT B 806 stab09 456CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:458U21 sense siNA BUAACCAUGAGGACCAGGUGTT B 807 stab09 590 UACCUGUGCACGUUGGAACUUUU 592CXCL12a:592U21 sense siNA B CCUGUGCACGUUGGAACUUTT B 808 stab09 807GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:809U21 sense siNA BUUAACAGGGAGCUGGAAAATT B 809 stab09 968 GGGCUCCAUGUAGAAGCCACUAU 594CXCL12a:970U21 sense siNA B GCUCCAUGUAGAAGCCACUTT B 810 stab09 991UACUGGGACUGUGCUCAGAGACC 595 CXCL12a:993U21 sense siNA BCUGGGACUGUGCUCAGAGATT B 811 stab09 1021 CAGCUAUUCCUACUCUCUCCCCG 596CXCL12a:1023U21 sense siNA B GCUAUUCCUACUCUCUCCCTT B 812 stab09 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1323U21 sense siNA BGUGACUGGGUUUUGUGAUUTT B 813 stab09 1340 UUGCCUCUGAAGCCUAUGUAUGC 598CXCL12a:1342U21 sense siNA B GCCUCUGAAGCCUAUGUAUTT B 814 stab09 190GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:210L21 antisense siNACAACAUGGCUUUCGAAGAATsT 815 (192C) stab10 213 AGAGCCAACGUCAAGCAUCUCAA 576CXCL12b:233L21 antisense siNA GAGAUGCUUGACGUUGGCUTsT 816 (215C) stab10214 GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:234L21 antisense siNAUGAGAUGCUUGACGUUGGCTsT 817 (216C) stab10 215 AGCCAACGUCAAGCAUCUCAAAA 578CXCL12b:235L21 antisense siNA UUGAGAUGCUUGACGUUGGTsT 818 (217C) stab10216 GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:236L21 antisense siNAUUUGAGAUGCUUGACGUUGTsT 819 (218C) stab10 217 CCAACGUCAAGCAUCUCAAAAUU 580CXCL12b:237L21 antisense siNA UUUUGAGAUGCUUGACGUUTsT 820 (219C) stab10218 CAACGUCAAGCAUCUCAAAAUUC 581 CXCL12b:238L21 antisense siNAAUUUUGAGAUGCUUGACGUTsT 821 (220C) stab10 219 AACGUCAAGCAUCUCAAAAUUCU 582CXCL12b:239L21 antisense siNA AAUUUUGAGAUGCUUGACGTsT 822 (221C) stab10998 CUCCACGUUCUGCUCAUCAUUCU 583 CXCL12b:1018L21 antisenseAAUGAUGAGCAGAACGUGGTsT 823 siNA (1000C) stab10 1000CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1020L21 antisenseAGAAUGAUGAGCAGAACGUTsT 824 siNA (1002C) stab10 1496CACUGCUUGAGGAAAACAAGCAU 585 CXCL12b:1516L21 antisenseGCUUGUUUUCCUCAAGCAGTsT 825 siNA (1498C) stab10 1821CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1841L21 antisenseUGCUUGCCUUUGUCAAAAGTsT 826 siNA (1823C) stab10 2109AAGGCAAACCCAUCAACAAAAAU 587 CXCL12b:2129L21 antisenseUUUUGUUGAUGGGUUUGCCTsT 827 siNA (2111C) stab10 2110AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2130L21 antisenseUUUUUGUUGAUGGGUUUGCTsT 828 siNA (2112C) stab10 2116ACCCAUCAACAAAAAUUGUCCCU 589 CXCL12b:2136L21 antisenseGGACAAUUUUUGUUGAUGGTsT 829 siNA (2118C) stab10 2631AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2651L21 antisenseGGACGCACUUGACUAGCAGTsT 830 siNA (2633C) stab10 456CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:476L21 antisense siNACACCUGGUCCUCAUGGUUATsT 831 (458C) stab10 590 UACCUGUGCACGUUGGAACUUUU 592CXCL12a:610L21 antisense siNA AAGUUCCAACGUGCACAGGTsT 832 (592C) stab10807 GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:827L21 antisense siNAUUUUCCAGCUCCCUGUUAATsT 833 (809C) stab10 968 GGGCUCCAUGUAGAAGCCACUAU 594CXCL12a:988L21 antisense siNA AGUGGCUUCUACAUGGAGCTsT 834 (970C) stab10991 UACUGGGACUGUGCUCAGAGACC 595 CXCL12a:1011L21 antisenseUCUCUGAGCACAGUCCCAGTsT 835 siNA (993C) stab10 1021CAGCUAUUCCUACUCUCUCCCCG 596 CXCL12a:1041L21 antisenseGGGAGAGAGUAGGAAUAGCTsT 836 siNA (1023C) stab10 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1341L21 antisenseAAUCACAAAACCCAGUCACTsT 837 siNA (1323C) stab10 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1360L21 antisenseAUACAUAGGCUUCAGAGGCTsT 838 siNA (1342C) stab10 190GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:210L21 antisense siNAcAAcAuGGcuuucGAAGAATT B 839 (192C) stab19 213 AGAGCCAACGUCAAGCAUCUCAA576 CXCL12b:233L21 antisense siNA GAGAuGcuuGAcGuuGGcuTT B 840 (215C)stab19 214 GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:234L21 antisense siNAuGAGAuGcuuGAcGuuGGcTT B 841 (216C) stab19 215 AGCCAACGUCAAGCAUCUCAAAA578 CXCL12b:235L21 antisense siNA uuGAGAuGcuuGAcGuuGGTT B 842 (217C)stab19 216 GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:236L21 antisense siNAuuuGAGAuGcuuGAcGuuGTT B 843 (218C) stab19 217 CCAACGUCAAGCAUCUCAAAAUU580 CXCL12b:237L21 antisense siNA uuuuGAGAuGcuuGAcGuuTT B 844 (219C)stab19 218 CAACGUCAAGCAUCUCAAAAUUC 581 CXCL12b:238L21 antisense siNAAuuuuGAGAuGcuuGAcGuTT B 845 (220C) stab19 219 AACGUCAAGCAUCUCAAAAUUCU582 CXCL12b:239L21 antisense siNA AAuuuuGAGAuGcuuGAcGTT B 846 (221C)stab19 998 CUCCACGUUCUGCUCAUCAUUCU 583 CXCL12b:1018L21 antisenseAAuGAuGAGcAGAAcGuGGTT B 847 siNA (1000C) stab19 1000CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1020L21 antisenseAGAAuGAuGAGcAGAAcGuTT B 848 siNA (1002C) stab19 1496CACUGCUUGAGGAAAACAAGCAU 585 CXCL12b:1516L21 antisenseGcuuGuuuuccucAAGcAGTT B 849 siNA (1498C) stab19 1821CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1841L21 antisenseuGcuuGccuuuGucAAAAGTT B 850 siNA (1823C) stab19 2109AAGGCAAACCCAUCAACAAAAAU 587 CXCL12b:2129L21 antisenseuuuuGuuGAuGGGuuuGccTT B 851 siNA (2111C) stab19 2110AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2130L21 antisenseuuuuuGuuGAuGGGuuuGcTT B 852 siNA (2112C) stab19 2116ACCCAUCAACAAAAAUUGUCCCU 589 CXCL12b:2136L21 antisenseGGAcAAuuuuuGuuGAuGGTT B 853 siNA (2118C) stab19 2631AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2651L21 antisenseGGAcGcAcuuGAcuAGcAGTT B 854 siNA (2633C) stab19 456CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:476L21 antisense siNAcAccuGGuccucAuGGuuATT B 855 (458C) stab19 590 UACCUGUGCACGUUGGAACUUUU592 CXCL12a:610L21 antisense siNA AAGuuccAAcGuGcAcAGGTT B 856 (592C)stab19 807 GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:827L21 antisense siNAuuuuccAGcucccuGuuAATT B 857 (809C) stab19 968 GGGCUCCAUGUAGAAGCCACUAU594 CXCL12a:988L21 antisense siNA AGuGGcuucuAcAuGGAGcTT B 858 (970C)stab19 991 UACUGGGACUGUGCUCAGAGACC 595 CXCL12a:1011L21 antisenseucucuGAGcAcAGucccAGTT B 859 siNA (993C) stab19 1021CAGCUAUUCCUACUCUCUCCCCG 596 CXCL12a:1041L21 antisenseGGGAGAGAGuAGGAAuAGcTT B 860 siNA (1023C) stab19 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1341L21 antisenseAAucAcAAAAcccAGucAcTT B 861 siNA (1323C) stab19 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1360L21 antisenseAuAcAuAGGcuucAGAGGcTT B 862 siNA (1342C) stab19 190GAUUCUUCGAAAGCCAUGUUGCC 575 CXCL12b:210L21 antisense siNACAACAUGGCUUUCGAAGAATT B 863 (192C) stab22 213 AGAGCCAACGUCAAGCAUCUCAA576 CXCL12b:233L21 antisense siNA GAGAUGCUUGACGUUGGCUTT B 864 (215C)stab22 214 GAGCCAACGUCAAGCAUCUCAAA 577 CXCL12b:234L21 antisense siNAUGAGAUGCUUGACGUUGGCTT B 865 (216C) stab22 215 AGCCAACGUCAAGCAUCUCAAAA578 CXCL12b:235L21 antisense siNA UUGAGAUGCUUGACGUUGGTT B 866 (217C)stab22 216 GCCAACGUCAAGCAUCUCAAAAU 579 CXCL12b:236L21 antisense siNAUUUGAGAUGCUUGACGUUGTT B 867 (218C) stab22 217 CCAACGUCAAGCAUCUCAAAAUU580 CXCL12b:237L21 antisense siNA UUUUGAGAUGCUUGACGUUTT B 868 (219C)stab22 218 CAACGUCAAGCAUCUCAAAAUUC 581 CXCL12b:238L21 antisense siNAAUUUUGAGAUGCUUGACGUTT B 869 (220C) stab22 219 AACGUCAAGCAUCUCAAAAUUCU582 CXCL12b:239L21 antisense siNA AAUUUUGAGAUGCUUGACGTT B 870 (221C)stab22 998 CUCCACGUUCUGCUCAUCAUUCU 583 CXCL12b:1018L21 antisenseAAUGAUGAGCAGAACGUGGTT B 871 siNA (1000C) stab22 1000CCACGUUCUGCUCAUCAUUCUCU 584 CXCL12b:1020L21 antisenseAGAAUGAUGAGCAGAACGUTT B 872 siNA (1002C) stab22 1496CACUGCUUGAGGAAAACAAGCAU 585 CXCL12b:1516L21 antisenseGCUUGUUUUCCUCAAGCAGTT B 873 siNA (1498C) stab22 1821CACUUUUGACAAAGGCAAGCACU 586 CXCL12b:1841L21 antisenseUGCUUGCCUUUGUCAAAAGTT B 874 siNA (1823C) stab22 2109AAGGCAAACCCAUCAACAAAAAU 587 CXCL12b:2129L21 antisenseUUUUGUUGAUGGGUUUGCCTT B 875 siNA (2111C) stab22 2110AGGCAAACCCAUCAACAAAAAUU 588 CXCL12b:2130L21 antisenseUUUUUGUUGAUGGGUUUGCTT B 876 siNA (2112C) stab22 2116ACCCAUCAACAAAAAUUGUCCCU 589 CXCL12b:2136L21 antisenseGGACAAUUUUUGUUGAUGGTT B 877 siNA (2118C) stab22 2631AACUGCUAGUCAAGUGCGUCCAC 590 CXCL12b:2651L21 antisenseGGACGCACUUGACUAGCAGTT B 878 siNA (2633C) stab22 456CUUAACCAUGAGGACCAGGUGUG 591 CXCL12a:476L21 antisense siNACACCUGGUCCUCAUGGUUATT B 879 (458C) stab22 590 UACCUGUGCACGUUGGAACUUUU592 CXCL12a:610L21 antisense siNA AAGUUCCAACGUGCACAGGTT B 880 (592C)stab22 807 GCUUAACAGGGAGCUGGAAAAAG 593 CXCL12a:827L21 antisense siNAUUUUCCAGCUCCCUGUUAATT B 881 (809C) stab22 968 GGGCUCCAUGUAGAAGCCACUAU594 CXCL12a:988L21 antisense siNA AGUGGCUUCUACAUGGAGCTT B 882 (970C)stab22 991 UACUGGGACUGUGCUCAGAGACC 595 CXCL12a:1011L21 antisenseUCUCUGAGCACAGUCCCAGTT B 883 siNA (993C) stab22 1021CAGCUAUUCCUACUCUCUCCCCG 596 CXCL12a:1041L21 antisenseGGGAGAGAGUAGGAAUAGCTT B 884 siNA (1023C) stab22 1321GAGUGACUGGGUUUUGUGAUUGC 597 CXCL12a:1341L21 antisenseAAUCACAAAACCCAGUCACTT B 885 siNA (1323C) stab22 1340UUGCCUCUGAAGCCUAUGUAUGC 598 CXCL12a:1360L21 antisenseAUACAUAGGCUUCAGAGGCTT B 886 siNA (1342C) stab22 Uppercase= ribonucleotide u,c = 2′-deoxy-2′-fluoro U,C T = thymidine B = inverteddeoxy abasic s = phosphorothioate linkage A = deoxy Adenosine G = deoxyGuanosine G = 2′-O-methyl Guanosine A = 2′-O-methyl Adenosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at 3′-ends S/AS “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All linkages Usually AS“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and 3′-ends — Usually S “Stab 5” 2′-fluoro Ribo — 1 at3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and 3′-ends — Usually S“Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′-ends — Usually S “Stab 8”2′-fluoro 2′-O-Methyl — 1 at 3′-end S/AS “Stab 9” Ribo Ribo 5′ and3′-ends — Usually S “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab11” 2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA5′ and 3′-ends Usually S “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo2′-O-Methyl 5′ and 3′-ends Usually S “Stab 17” 2′-O-Methyl 2′-O-Methyl5′ and 3′-ends Usually S “Stab 18” 2′-fluoro 2′-O-Methyl 5′ and 3′-endsUsually S “Stab 19” 2′-fluoro 2′-O-Methyl 3′-end S/AS “Stab 20”2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and 3′-ends Usually S “Stab 24” 2′-fluoro* 2′-O-Methyl* — 1at 3′-end S/AS “Stab 25” 2′-fluoro* 2′-O-Methyl* — 1 at 3′-end S/AS“Stab 26” 2′-fluoro* 2′-O-Methyl* — S/AS “Stab 27” 2′-fluoro*2′-O-Methyl* 3′-end S/AS “Stab 28” 2′-fluoro* 2′-O-Methyl* 3′-end S/AS“Stab 29” 2′-fluoro* 2′-O-Methyl* 1 at 3′-end S/AS “Stab 30” 2′-fluoro*2′-O-Methyl* S/AS “Stab 31” 2′-fluoro* 2′-O-Methyl* 3′-end S/AS “Stab32” 2′-fluoro 2′-O-Methyl S/AS “Stab 33” 2′-fluoro 2′-deoxy* 5′ and3′-ends — Usually S “Stab 34” 2′-fluoro 2′-O-Methyl* 5′ and 3′-endsUsually S “Stab 3F” 2′-OCF3 Ribo — 4 at 5′-end Usually S 4 at 3′-end“Stab 4F” 2′-OCF3 Ribo 5′ and 3′-ends — Usually S “Stab 5F” 2′-OCF3 Ribo— 1 at 3′-end Usually AS “Stab 7F” 2′-OCF3 2′-deoxy 5′ and 3′-ends —Usually S “Stab 8F” 2′-OCF3 2′-O-Methyl — 1 at 3′-end S/AS “Stab 11F”2′-OCF3 2′-deoxy — 1 at 3′-end Usually AS “Stab 12F” 2′-OCF3 LNA 5′ and3′-ends Usually S “Stab 13F” 2′-OCF3 LNA 1 at 3′-end Usually AS “Stab14F” 2′-OCF3 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 15F”2′-OCF3 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 18F” 2′-OCF32′-O-Methyl 5′ and 3′-ends Usually S “Stab 19F” 2′-OCF3 2′-O-Methyl3′-end S/AS “Stab 20F” 2′-OCF3 2′-deoxy 3′-end Usually AS “Stab 21F”2′-OCF3 Ribo 3′-end Usually AS “Stab 23F” 2′-OCF3* 2′-deoxy* 5′ and3′-ends Usually S “Stab 24F” 2′-OCF3* 2′-O-Methyl* — 1 at 3′-end S/AS“Stab 25F” 2′-OCF3* 2′-O-Methyl* — 1 at 3′-end S/AS “Stab 26F” 2′-OCF3*2′-O-Methyl* — S/AS “Stab 27F” 2′-OCF3* 2′-O-Methyl* 3′-end S/AS “Stab28F” 2′-OCF3* 2′-O-Methyl* 3′-end S/AS “Stab 29F” 2′-OCF3* 2′-O-Methyl*1 at 3′-end S/AS “Stab 30F” 2′-OCF3* 2′-O-Methyl* S/AS “Stab 31F”2′-OCF3* 2′-O-Methyl* 3′-end S/AS “Stab 32F” 2′-OCF3 2′-O-Methyl S/AS“Stab 33F” 2′-OCF3 2′-deoxy* 5′ and 3′-ends — Usually S “Stab 34F”2′-OCF3 2′-O-Methyl* 5′ and 3′-ends Usually S CAP = any terminal cap,see for example FIG. 10. All Stab 00-34 chemistries can comprise3′-terminal thymidine (TT) residues All Stab 00-34 chemistries typicallycomprise about 21 nucleotides, but can vary as described herein. S =sense strand AS = antisense strand *Stab 23 has a single ribonucleotideadjacent to 3′-CAP *Stab 24 and Stab 28 have a single ribonucleotide at5′-terminus *Stab 25, Stab 26, and Stab 27 have three ribonucleotides at5′-terminus *Stab 29, Stab 30, Stab 31, Stab 33, and Stab 34 any purineat first three nucleotide positions from 5′-terminus are ribonucleotidesp = phosphorothioate linkage

TABLE V A. 2.5 μmol Synthesis Cycle ABI 394 Instrument ReagentEquivalents Amount Wait Time* DNA Wait Time* 2′-O-methyl Wait Time*RNAPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Reagent Equivalents AmountWait Time* DNA Wait Time* 2′-O-methyl Wait Time*RNA Phosphoramidites 1531 μL 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min465 sec Acetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124μL 5 sec 5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 secIodine 20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle96 well Instrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* 2′-O-Reagent 2′-O-methyl/Ribo methyl/Ribo Wait Time* DNA methyl Wait Time*Ribo Phosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 secS-Ethyl Tetrazole  70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

1. A chemically modified, short interfering nucleic acid (siNA)molecule, wherein: (a) the siNA comprises a sense strand and a separateantisense strand, each strand having one or more pyrimidine nucleotidesand one or more purine nucleotides; (b) each strand is independently 18to 27 nucleotides in length, and together comprise a duplex havingbetween 17 and 23 base pairs; (c) the antisense strand is complementaryto a human stromal cell-derived factor-1 (SDF-1) RNA sequence comprisingSEQ ID NO: 907; and, (d) a plurality of the pyrimidine nucleotidespresent in the sense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and a plurality of the purine nucleotides present in thesense strand are 2′-deoxy purine nucleotides. 2-13. (canceled)
 14. ThesiNA of claim 1, wherein the sense strand includes a terminal cap moietyat both 5′ and 3′-ends. 15-18. (canceled)
 19. The siNA of claim 1,wherein the sense strand, the antisense strand, or both the sense strandand the antisense strand include a 3′-overhang.
 20. (canceled)
 21. ThesiNA molecule of claim 1, wherein a plurality of the pyrimidinenucleotides in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and a plurality of the purine nucleotides present in theantisense strand are 2′-O-methyl purine nucleotides.
 22. The siNA ofclaim 21, wherein the antisense strand has a phosphorothioateinternucleotide linkage at the 3′ end.
 23. The siNA molecule of claim 1,wherein a plurality of the pyrimidine nucleotides present in theantisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and aplurality of the purine nucleotides present in the antisense strand are2′-deoxy purine nucleotides.
 24. The siNA molecule of claim 23, whereinthe antisense strand has a phosphorothioate internucleotide linkage atthe 3′ end.
 25. A composition comprising the siNA molecule of claim 1and a pharmaceutically acceptable carrier or diluent.